K. B. Zwart

Methanobacterium formicicum, an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

The Gram-positive methanogenic endosymbiont of the sapropelic ciliateMetopus striatus was isolated and identified asMethanobacterium formicicum. In the ciliate cell the methanogens are in close association with microbody-like organelles. No mitochondria could be detected. The nature of the microbodies and the physiological background of the observed association are discussed.

Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 μm. It had a generation time of 7 or 12 hours on growth on H2/CO2 or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.

Application of rumen microorganisms for an enhanced anaerobic degradation of solid organic waste materials

Abstract Organic waste materials of various origin and composition were screened for anaerobic digestion by a mixed population of rumen microorganisms. High loading rates could be applied, varying between 17·1 (coffee pulp) and 34·4 g (papermill sludge) of Volatile Solids per litre per day (VS litre−1 day−1) at retention times of only 60 h. Degradation efficiencies obtained for most waste materials were in the range of 60–70%, irrespective of their chemical composition. During the degradation of lignocellulosic wastes an apparent lignin degradation up to 48% was observed. Average production of Volatile Fatty Acids and biogas per gram of VS digested varied between 6·6 and 8·2 mmoles and 0·18 and 0·29 litre (approximately 40% CH4), respectively, for most wastes. Molar proportions of butyrate were higher with substrates rich in cell solubles, as compared to cellulosic and lignocellulosic substrates which yielded more propionate. Rumen ciliates belonging to the Entodinium group were predominant during the fermentation of substrates rich in cell solubles, whereas ciliates of the Diplodinium group were predominant with cellulosic and lignocellulosic substrates.

Symbiosis of methanogenic bacteria and anaerobic ciliates

were performed as described previously (Van Beelen et al., 1983). A typical mixture consisted of 40 nmol methenyt-THMP, 10 gmol 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, and 0.5 to 5 pt of cell-free extract (about 5 mg proteinml ~) in a total volume of 250 gt. The mixtures were incubated under an atmosphere of 80~ H2/20~ COz at 98 kPa for 30 rain at 60°C. The reaction was stopped by addition of 250 pl oxygen-free methanol. Methenyl-THMP was readily converted under these conditions and two products, detectable by HPLC analysis at 240 rim, were formed. The major product showed a retention time of 9 rain while the minor product appeared at 10.5 min. These compounds may be 5,10-methylene-5,6,7,8-tetrahydromethanopterin, 5or 10-methenyl-5,6,7,8-tetrahydromethanopterin or 5,6,7,8-tetrahydromethanopterin which are probably the other methanopterin derivatives involved in the methanogenic pathway from CO2 (Vogels and Visser, 1983).

Episymbiotic association of methanogenic bacteria and rumen ciliates

Methane fermentation is a useful process for liquid wastewater treatment because it reduces the chemical oxygen demand before disposal and yields the valuable fuel methane. Although most of the problems concerning the hardware technology for full-scale application of this fermentation process have been overcome, understanding of the microbial basis of methanogenie fermentation of wastes should facilitate process control and improve reactor performance. Since methanogenic bacteria contain a number of unique cofactors, it might be possible to use these compounds as specific parameters for the identification and quantitation of methanogens in anaerobic sludge. The presence ofcoenzyme F4zo allows the identification ofmethanogenic bacteria in mixed populations by epifluorescenee microscopy; in this way information is obtained concerning the location and interactions of methanogens. Chromatographic analysis of coenzyme F4z0 derivatives, which differ in the number of glutamate groups present in the molecule, yields information on the presence of Methanosarcina species among the methanogens. Since an aberrant corrinoid and a specific group of 7-methylpterins, among which methanopterin, are present in methanogens, the analysis of such compounds is now successfully used in studies on the activity ofmethanogens in various anaerobic reactors.

Improved anaerobic conversion of lignocellulosic waste using rumen microorganisms

By the discovery and structural elucidation of methanofuran, methanopterin, coenzyme M and factor F~s o the route of reduction of CO2 to methane appears to be roughly established (see for reviews: Daniels et al., 1984; Keltjens, 1984; Vogels et al., 1986), though a number of questions remain to be answered. Least is known about the first step of reduction of COz resulting in formylmethanofuran as the first detectable intermediate (Leigh, 1983). This endothermic reaction appears to be intimately coupled to the exothermic final steps of reduction by a phenomenon known as the RPG-effect. Our experiments ruled out the possibility that this coupling is a matter of consumption and production of energy in the first and terminal step, respectively. The formyl group bound to methanofuran is transferred to tetrahydromethanopterin (H4MPT) in an ATP-independent reaction resulting into the formation of 10-formyl-HgMPT that is subsequently converted to 5,10-methenyl-H4MPT by a cyclohydrolase. The terminal step of methanogenesis, the reductive demethylation of methylcoenzyme M is catalysed by a complex system of enzymes and the coenzymes component B and ATP. The coenzymes are involved in an activation process. Component B, which is an adenosine derivative, that may contain coenzyme M, can replace ATP at the activation, whereas ATP in the presence ofmethylcoenzyme M can replace component B.