Proc. Natl. Acad. Sci. USA Vol. 76, No. 1, pp. 494-498, January 1979 Microbiology Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri (methanogenesis/choline and betaine fermentation/creatine/sarcosine/growth yields) H. HIPPE*, D. CASPARIt, K. FIEBIGt, AND G. GOTTSCHALKt tInstitut fur Mikrobiologie der Universitat Gottingen and *Deutsche Sammiung von Mikroorganismen der Gesellschaft fur Strahlen- und Umweltforschung mbH Munchen, Grisebachstrasse 8, D-3400 Gottingen, West Germany Communicated by H. A. Barker, October 2, 1978 ABSTRACT A number of N-methyl compounds, including pounds including the monomethylamine compounds sarcosine several methylamines, creatine, sarcosine, choline, and betaine, and creatine were tested for their ability to serve as substrates were readily fermented by enrichment cultures yielding methane as a major product. Methylamine, dimethylamine, in methanogenesis. The results reported here demonstrate that trimeth lamine, and ethyldimethylamine were fermented by the methylamines are used for methane formation by pure pure cultures of Methanosarcina barkeri; except for ethyldi- cultures of M. barkeri and that other N-methyl compounds are methylamine, these amines are considered important substrates readily fermented to methane by mixed cultures. of this methanogenic microorganism. Creatine, sarcosine, choline, and betaine were fermented to methane only by mixed METHODS AND MATERIALS cultures. During growth of M. barkeri on methyl-, dimethyl-, or trimethylamine, methanol was not excreted into the medium. Organisms. Cultures of M. barkeri strain MS (neotype strain) The fermentation of trimethylamine gave rise to an interme- (DSM 800), Fusaro (DSM 804), 3 (DSM 805), and "Zhilina" diary accumulation of methyl- and dimethylamine in the me- (DSM 1232) were obtained from the German Collection of dium. An accumulation of methylamine during the fermenta- Microorganisms, Gottingen. Strain Kolksee was isolated from tion of dimethylamine was not observed. Methane and ammo- nia were produced from the three methylamines by M. barkeri a methanogenic enrichment culture of Lake Kolksee, Germany, in amounts expected on the basis of the appropriate fermenta- mud and calcium acetate. Strain G-1 was obtained from M. P. tion equations. The growth yield was 5.8 mg of cells (dry weight) Bryant, Urbana, IL, and has been isolated from anaerobic per mmol of methane and was not dependent on the kind of sewage sludge from the sewage plant at Gottingen, Germany. methyl compound used as substrate. Cultures of Methanobacterium ruminantium PS (DSM 861) and Mb. strain MOH(DSM 863) were received from K. Braun, The methanogenic bacteria are very restricted with respect to Institute of Microbiology, Gottingen. the nature of the substrates that can be used for methane for- Growth Media. The Hungate technique (18, 19) for media mation. All species described are able to produce methane from preparation and cultivation, as modified by Bryant (20), was molecular hydrogen and carbon dioxide. In addition, some used. The media were prepared under an atmosphere of 80% species can use carbon monoxide, formate, acetate, or methanol nitrogen/20% carbon dioxide; traces of oxygen were removed as a methanogenic substrate (1-5). Among this group of highly by passing the gas mixture through a heated copper column. specialized organisms Methanosarcina barkeri seems to be most The Methanosarcina strains were cultivated in a medium versatile with respect to the number of substrates utilized. It is (pH 6.5-6.8) that contained the following (per liter): K2HPO4, able to use all the compounds mentioned above, except formate, 0.348 g; KH2PO4, 0.227 g; NH4Cl, 0.5 g; MgSO4-7H20, 0.5 g; for methane formation (1, 6, 7). CaCl2-2H20, 0.25 g; NaCl, 2.25 g; FeSO4*7H20, 2 mg; resa- In addition to methanol, a rather large group of compounds zurin, 1 mg; vitamin solution (21), 10 ml; trace elements solution exists containing methyl groups which can be transferred easily (22) without Na2 EDTA and FeSO4, 3 ml; yeast extract (Difco), to other acceptors or carriers: the N-methyl compounds. Little 2 g; casitone (Difco), 2 g; NaHCO3, 0.85 g; methanol, 10 ml; is known about their role in methanogenesis. Zhilina and Zav- cysteine hydrochloride, 0.3 g; and Na2S-9H20, 0.3 g. Methanol arzin (8) reported growth on methylamine of a mixed culture (50% vol/vol) and the reducing agents were heat-sterilized of Methanosarcina and Desulfovibrio. Recently, it has been separately under N2 as concentrated aqueous solutions. Neutral demonstrated that the methyl groups of choline are converted stock solutions of amino compounds were filter-sterilized and to methane via trimethylamine in the rumen (9). Methyl- and freed from with 80% The dimethylamine are naturally occurring compounds (10, 11) but oxygen by flushing N2/20% CO2. probably trimethylamine is more abundant in anaerobic hab- substrate solutions were injected into the autoclaved medium itats. It is formed from choline by organisms such as Desulfo- with a hypodermic syringe prior to the addition of the reducing vibrio desulfuricans (12, 13). Furthermore, the trimethyl- agents. The final concentration of the amino compounds in the amine-N-oxide present in marine fish is reduced by bacteria medium was 50 or 100 mM (see Results). to trimethylamine after the fish have died (14). Several bacteria, For enrichments, the same medium but without yeast extract, especially enterobacteria and phototrophic bacteria, have been casitone, and cysteine was used. For some enrichments (see shown to carry out this reduction under anaerobic conditions Table 1), the medium of Barker (23) was used, which contained (15-17). Another potential precursor of trimethylamine is be- per liter of tap water the following minerals: K2HPO4, 0.4 g (pH is un- 6.8); NH4Cl, 1.0 g; MgCl2, 0.1 g; and Na2S-9H20, 0.3 g. taine, the fate of which under anaerobic conditions Methanobacterium ruminantium and Mb. strain MOH known. were grown in the following medium (grams per liter): Trimethylamine and a number of other N-methyl com- KH2PO4, 0.5 g; MgSO4-7H20, 0.4 g; NaCl, 0.4 g; NH4Cl, 0.4 g; CaCl2.2H20, 0.05 g; FeSO4.7H20, 2 mg; trace elements so- The publication costs of this article were defrayed in part by page lution 2 vitamin solution 10 1 charge payment. This article must therefore be hereby marked "ad- (21), ml; (21), ml; resazurin, mg; vertisement" in accordance with 18 U. S. C. §1734 solely to indicate sodium acetate, 1 g; yeast extract (Difco), 2 g; cysteine hydro- this fact. chloride, 0.5 g; Na2S-9H20, 0.5 g; and NaHCO3, 4.0 g (pH 7.0). 494 Downloaded by guest on September 28, 2021 Microbiology: Hippe et al. Proc. Natl. Acad. Sci. USA 76 (1979) 495 The gas atmosphere was 80% H2/20% CO2. For testing utili- resuspended in 0.5 ml of 1 M NaOH. After 10 min at room zation of methylamines, the medium was supplemented by 25 temperature, the alkaline suspension was heated for 15 sec at mM amine, and a gas mixture of 80% N2 and 20% CO2 was 100°C and thereafter quickly cooled in an ice bath. The sample used. was centrifuged and 20-100 ,ul of the supernatant was used for Cultivation and Enrichment Methods. Enrichments were protein determination. The aliquots were made up to 100,u made in tubes (Bellco 18 X 142 mm or anaerobic culture tubes, final volume with 1 M NaOH when necessary, and 5 ml of Hungate type) that contained 10 ml of medium. They were Coomassie brilliant blue (0.01%) was added. After 5 min at inoculated with 1 ml of anaerobic mud samples and incubated room temperature the absorption at 595 nm was measured, and at 30'C. Pure cultures of Methanosarcina were grown in S ml the protein concentration was read from a calibration curve of medium in similar tubes at 300C; they were maintained by made from standard solutions of bovine serum albumin in 1 M transfer of 5% inocula. Stock cultures were kept on methanol NaOH. Linearity was found in the range from 5 to 45 ,ug of medium at room temperature in order to extend the interval protein per 100-,ul sample. between transfers. We frequently checked cultures for purity Determination of cell dry weight was performed by filtering by inoculating CMC medium (24), medium for sulfate reducers 10- to 20-ml culture samples through preweighed membrane (25), and AC medium (Difco) and by microscopic observa- filters (SM 11303, 1.2-jum pore size, Sartorius, Gottingen, Ger- tion. many). After they were washed with 0.9% NaCl, the filters were For growth studies on methylamines, 100 ml of medium was dried to constant weight at 95°C and reweighed against con- prepared in heavy-walled 1-liter bottles closed with black trols. Ammonia was determined enzymatically with glutamate rubber stoppers, which were held in place by a wire closure dehydrogenase (28). during autoclaving and culturing. Enzymes and Chemicals. Glutamate dehydrogenase (EC Analytic Methods. Methylamines and methanol were de- 1.4.1.3), ADP, and NADPH were obtained from Boehringer termined by gas chromatography using a Perkin Elmer Model Mannheim, Germany. 2-Keto-glutaric acid was from Schu- 900, equipped with a 6 m X 2 mm glass column, packed with chardt, Munchen; Coomassie brilliant blue G-250 and bovine Pennwalt 231 GC-Packing (Applied Science Laboratories, Inc., serum albumin were from Serva, Heidelberg, Germany. Me- State College, PA), and with a flame ionization detector. The thylamine hydrochloride, dimethylamine hydrochloride, tri- injector temperature was 2250C, the oven temperature was methylamine hydrochloride, betaine, choline hydrochloride, 550C, and the detector temperature was 250°C. Nitrogen was creatine, sarcosine, N,N-dimethylformamide, and N,N-di- used as carrier gas at a flow rate of 20 ml min-1. Before injec- methylurea were purchased from Merck, Darmstadt, Germany.