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Three novel halotolerant, hydrogenotrophic methanogens, designated strains K1F9705bT, K1F9705c and O1F9704a, were isolated from an estuary in Eriln Shi, Taiwan, and from a nearby marine water aquaculture fishpond. These isolates were irregular cocci that stained Gram-negative. Strains K1F9705bT and K1F9705c were non-motile, but strain O1F9704a was weakly motile with flagella. They were able to use formate and H2/CO2 to form methane, but they could not catabolize acetate, methanol, trimethylamine or secondary alcohols. Acetate was required for cell growth. Tungsten greatly stimulated the growth of strains K1F9705bT and K1F9705c, but did not affect the growth of strain O1F9704a. Optimal pH and temperature for growth of these three isolates were respectively 7.2 and 37 degrees C. Optimal NaCl concentration for growth was 0.5% for strain O1F9704a and 1.0% for strains K1F9705c and K1F9705bT. Moreover, all strains grew well at up to 8-12% NaCl. Analysis of the 16S rRNA gene revealed that these isolates are members of the genus Methanocalculus, but are distinct from Methanocalculus taiwanensis, Methanocalculus pumilus and Methanocalculus halotolerans, with sequence similarities of 98.4, 98.3 and 98.2%, respectively. In addition, strain K1F9705bT possessed 85, 80, 37, 29 and 10% DNA-DNA relatedness to strain K1F9705c, strain O1F9704a, M. pumilus, M. halotolerans and M. taiwanensis, respectively. Analysis of protein profiles and the Mr of surface (S)-layer glycoprotein subunits showed that these three new isolates are closely related to, but distinct from, known Methanocalculus species. A novel species, Methanocalculus chunghsingensis sp. nov., is proposed for strains K1F9705bT, K1F9705c and O1F9704a. The type strain is K1F9705bT (=OCM 772T=DSM 14646T).

Methanocalculus chunghsingensis sp. nov., isolated from an estuary and a marine fishpond in Taiwan