Eur J App! Microbiol Biotechnol (1983) 18 : 67-69 Short Communication European ~o~ro~Appiled Microbiology and Biotechnology 9 Springer-Verlag 1983 Quantitation of Coenzyme F420 in Methanogenic Sludge by the Use of Reversed-Phase High-Performance Liquid Chromatography and a Fluorescence Detector P. van Beelen, A. C. Dijkstra, and G. D. Vogels Department of Microbiology, Faculty of Science, University of Ni]megen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands SUI~IARY. with the use of acetone extraction, reversed-phase High-Performance Liquid Chromatography and fluorimetric monitoring, the quantity of coenzyme F420 in mixed liquors and rumen contents can be measured. A synthetic analog of coenzyme F420 is used as an internal standard to compensate for differences in fluorimetric monitoring. The method allows the detection of one picomol of coenzyme F420 and the differentiation between different forms of the coenzyme known to be present in various methanogenic bacteria. INTRODUCTION Anaerobic digestion is becoming increasingly important for the purification of wastewaters rich in organic substances. Methanogenesis is thought to be often the rate-limiting step in this process. Measurement of the eoenzyme F~20 content of mixed liquors from anaerobic digestors by fluorimetric monitoring was used by Delafontaine et el. (1979) to establish the potential methanogenic activity of the liquor. Coenzyme F420 was identified as the N-(N-L-lactyl-y-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy- 5-deazariboflavin 5'-phosphate (Eirich et el. 1978). It is present in all methanogenic bacteria tested (Eirich et el. 1979) and it functions as an electron carrier in methanogenesis and cell carbon synthesis (Vogels et el. 1982). Since substances which absorb light at 420 or 470 nm will interfere with the direct measurement of coenzyme F42G, we introduce here a method by which coenzyme F420 is first separated by reversed-phase High-Performance Liquid Chromatography (HPLC), which is known to separate coenzymes from methanogenic bacteria (Van Beelen et al. 1983), and then measured by fluorimetric monitoring. An internal standard with the same fluorimetric properties as coenzyme F420, but with a different retention time, is added to the samples to compensate for differences in fluorimetric monitoring and for changes in the volume during sample preparation. MATERIALS AND METHODS Microorganisms. Methanobacterium thermoauto- trophicum strain AH, was grown according to Sch@nheit et al. (1979). Methanosaroi~a barkeri, Methanococcus mazei and the unidentified strain CSM 16 (all grown in a medium containing methanol (!0 ml/l), Feast extract (2 g/l), trypto~ 8oya broth (2 g/l), buffer, minerals, vitamins, reducing agents and a gas phase of 80% H 2 and 20% C02) were gifts of J.J.A. van Bruggen. Strain CSM 16 was isolated from the digestor fed with beet sugar waste (Table I). Ckemioals. 7,8-Didemethyl-8-hydroxy-5- deazariboflavin (FO) was prepared according to Ashton et el. (1979). Coenzyme F420 was pmrified from M. thermoautotrophicum according to Eirich et el. (1978). Sample preparation. FO was added to at least 20 mg of mixed liquor to obtain a final concentration of 4 ~M. This mixture was boiled for 5 min. One volume of acetone was added after cooling and the mixture was vigorously shaken and centrifuged for i0 min at 9,700 g. A sample of 25 ~i of the supernatant was brought into the HPLC injector. Cells obtained from i0 ml culture medium were suspended in 0.25 ml glass distilled water and treated as described above. Storage of mixtures which contained either FO or Offprint requests to." P. van Beelen