Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Fatty acids and alcohols are key intermediates in the methanogenic degradation of organic matter, e.g., in anaerobic sewage sludge digestors or freshwater lake sediments. They are produced by classical fermenting bacteria for disposal of electrons derived in simultaneous substrate oxidations. Methanogenic bacteria can degrade primarily only one-carbon compounds. Therefore, acetate, propionate, ethanol, and their higher homologs have to be fermented further to one-carbon compounds. These fermentations are called secondary or syntrophic fermentations. They are endergonic processes under standard conditions and depend on intimate coupling with methanogenesis. The energetic situation of the prokaryotes cooperating in these processes is problematic: the free energy available in the reactions for total conversion of substrate to methane attributes to each partner amounts of energy in the range of the minimum biochemically convertible energy, i.e., 20 to 25 kJ per mol per reaction. This amount corresponds to one-third of an ATP unit and is equivalent to the energy required for a monovalent ion to cross the charged cytoplasmic membrane. Recent studies have revealed that syntrophically fermenting bacteria synthesize ATP by substrate-level phosphorylation and reinvest part of the ATP-bound energy into reversed electron transport processes, to release the electrons at a redox level accessible by the partner bacteria and to balance their energy budget. These findings allow us to understand the energy economy of these bacteria on the basis of concepts derived from the bioenergetics of other microorganisms.

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Energetics of syntrophic cooperation in methanogenic degradation.

A new hypothesis for the origin of eukaryotic cells is proposed, based on the comparative biochemistry of energy metabolism. Eukaryotes are suggested to have arisen through symbiotic association of an anaerobic, strictly hydrogen-dependent, strictly autotrophic archaebacterium (the host) with a eubacterium (the symbiont) that was able to respire, but generated molecular hydrogen as a waste product of anaerobic heterotrophic metabolism. The host's dependence upon molecular hydrogen produced by the symbiont is put forward as the selective principle that forged the common ancestor of eukaryotic cells.

The hydrogen hypothesis for the first eukaryote

In addition to the bacteria in the rumen there are many larger (5–250/mm long) organisms which at various times have been designated protozoa Of these the ‘ovals’ (Quin’s and Eadie’s) are now known to be large bacteria (Orpin, 1976) and the ‘flagellates’ Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis are the zoospores of phycomycete fungi (Orpin, 1977a, b) There are genuine flagellates in the rumen, eg Trichomonas spp, Monoceromonas sp and Chilomastix sp, but almost nothing is known about their metabolism (Jensen and Hammon, 1964) The largest, most obvious and most important protozoa are the ciliates, of which there are two groups both in the subclass Trichostomatia The so called ‘holotrich’ protozoa belong to the order Vestibuliferida and the entodiniomorphs to the order Entodiniomorphida, suborder Entodiniomorphina and family Ophryoscolecidae As the properties and metabolism of these two protozoal groups are different, they will be considered separately below

The Rumen Protozoa

Kingdom protozoa and its 18 phyla.

Fluorescent bacteria were demonstrated to be abundantly spread as single cells throughout the cytoplasm of the giant amoeba Pelomyxa palustris, the sapropelic ciliate Metopus striatus and six other anaerobic protozoa examined. The endosymbionts of P. palustris and M. striatus were identified as methanogenic bacteria on the basis of the presence of the deazaflavin coenzyme F420 and the pterin compound F342. Moreover individuals of P. palustris produced methane over a long period of incubation. The number of methanogenic bacteria was above 1010 cells/ml protozoal cytoplasm. Two types of methanogenic bacteria together with unidentified thick bacteria were found in P. palustris. The physiological background of this endosymbiosis and its functioning in degradation processes in the anoxic environment are discussed.

Symbiosis of methanogenic bacteria and sapropelic protozoa

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.

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

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.

Methanobacterium formicicum, an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

Psalteriomonas lanterna gen. nov., sp. nov., a free-living amoeboflagellate isolated from freshwater anaerobic sediments

~~~~~~~~~~~~~~ A cytochemical staining method was developed for the demonstration of hydrogenase activity in hydrogenosomes of Trichomonas vaginalis. After fixation in low concentrations of glutaraldehyde under strictly anaerobic conditions and incubation of these cells in the presence of H2 and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. The method was used to demonstrate the hydrogenosomal nature of similar microbodies of the sapropelic ciliates Plagiopyla nasuta and Trimyema compressum.

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Claudius K. Stumm

Papers

Symbiosis of methanogenic bacteria and sapropelic protozoa

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

Methanobacterium formicicum, an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

Psalteriomonas lanterna gen. nov., sp. nov., a free-living amoeboflagellate isolated from freshwater anaerobic sediments

Cytochemical Localization of Hydrogenase Activity in the Anaerobic Protozoa Trichomonas vaginalis, Plagiopyla nasuta and Trimyema compressum