![]() Freeze etching was carried out as described previously (Rachel et al. Samples on grids were then either negatively stained for 1 min with 2% uranyl acetate (w/v) or shadowed with Pt/C (15° angle CFE 50 Cressington). 10 µl of cell concentrate were placed on hydrophilized 400-mesh carbon-coated copper grids (Plano). ![]() Gram-stain was carried out as described previously (Boone and Whitman 1988) using Escherichia coli K12 (ATCC ® 25922 ™) and Bacillus atrophaeus (ATCC ® 9372 ™) as reference strains.įor electron microscopy, cells in late exponential growth phase were fixed with 1% glutardialdehyde (final concentration v/v) for 10 min at room temperature and concentrated by centrifugation (4000× g, 15 min). 2014) connected to a phase-contrast microscope (Olympus BX53). Motility was surveyed at 37 ☌ under anaerobic conditions using a temperature gradient-forming device (Mora et al. Light microscopy was performed using a phase-contrast and fluorescence microscope (Olympus BX60 Bright Line HC 434/17 excitation filter with a beam splitter 452 nm and long pass filter U-E455). Logarithmic cell cultures were stored at 4 ☌ for 2–3 months for short-term storage. For long-term conservation, cells were anaerobically centrifuged (3000× g, 30 min), resuspended in medium containing 5% DMSO, sealed in glass capillaries and stored over liquid nitrogen in our in-house culture collection. ![]() For further studies, acetate supplement was reduced from 0.5 to 0.1%. After two transfers, single cell isolation was performed using an optical tweezer setup (Huber et al. Initial cell growth with irregular cocci occurred after 1 week of incubation at 37 ☌ with shaking. Inoculation was performed with 0.5 ml of original samples in 20 ml medium supplemented with 0.5% acetate (w/v) and H 2/CO 2 as gas phase. 20 ml medium were aliquoted into 120 ml serum bottles and pressurized with H 2/CO 2 (80:20, v/v, 300 kPa) or N 2/CO 2 (80:20, v/v, 200 kPa), respectively. It was reduced with 0.5 g Na 2S × 2–3 H 2O and the pH was adjusted to 6.5 with 1 M HCl. The medium was prepared according to the standard techniques for anaerobic cultivation (Balch and Wolfe 1976). 1979) was used for enrichment and cultivation of strain CaP3V-MF-L2A T. MS medium, modified from Balch’s medium I (Balch et al. Water samples were taken under sterile and anaerobic conditions in a depth of 20–30 cm sub-watersurface using 100 ml glass bottles. Strain CaP3V-MF-L2A T was isolated from an exploratory oil well in Cahuita National Park, located at the southwestern Atlantic coast of Costa Rica (SI Fig. Therefore, we propose the here described strain CaP3V-MF-L2A T as a novel species, Methanofollis propanolicus sp. ![]() Furthermore, strain CaP3V-MF-L2A T is characterized by a smaller cell size, its motility and a significantly shorter generation time. Strain CaP3V-MF-L2A T cannot grow on ethanol or 1-butanol but utilizes 1-propanol and 2-propanol for methanogenesis. With the increasing number of described species, it became clear that the genus’ capability to use primary and secondary alcohols with two or more C-atoms for methane production is a characteristic feature.ĬaP3V-MF-L2A T shows a similarity of 98.8% in 16S rRNA gene sequence towards Methanofollis ethanolicus however, their physiological characteristics differ significantly. 2009) was discovered in a lotus field, while the recently described Methanofollis fontis was isolated from a marine sediment near a cold seep (Chen et al. 2005) and Methanofollis aquaemaris (Lai and Chen 2001) were both isolated from a fish pond in Taiwan. Methanofollis liminatans was derived from a wastewater reactor in Germany and Methanofollis tationis from a solfataric field in Chile. These species were isolated from various aquatic environments with different salinities. Today, six validly described species are assigned to the genus Methanofollis. Furthermore, Methanofollis tationis possesses a pterin that is different from the known methanopterin or sarcinapterin–tatiopterin has not been found in any other methanogen since then (Raemakers-Franken et al. liminatans were transferred to the novel genus Methanofollis due to distinct patterns of glycolipids, phosphoglycolipids and amino-phosphoglycolipids among others. With the publication of this genus, the former Methanogenium species M. 1999) represents one out of six genera within the family Methanomicrobiaceae (Balch et al.
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