A novel methanogen, designated strain N2F9704T, was isolated from an aquaculture fish pond near Wang-gong, Taiwan. The cells were irregular cocci, non-motile, 1.2-2.0 microm in diameter and stained gram-negative. Cells of strain N2F9704T lysed easily by SDS treatment (0.1 g l(-1)) and the S-layer protein had an Mr of 137000. The catabolic substrates used included formate and H2+CO2, but not acetate, methanol, trimethylamine or secondary alcohols. The optimal growth parameters for strain N2F9704T were pH 6.5, 37 degrees C with 0.5% NaCl. Trace amounts of tungstate not only promoted growth but also extended the range of growth conditions. Analysis of the 16S rDNA sequence revealed a phylogenetic relationship to Methanofollis species and the name Methanofollis aquaemaris sp. nov. is therefore proposed for strain N2F9704T (= OCM 746T = CCRC 16166T). Additionally, the strain was infected with a novel coccus-shaped, enveloped virus with a diameter of 200 nm.

A mesophilic, hydrogenotrophic methanogen, strain S3Fa^T, was isolated from sediments collected by Ocean Researcher I cruise ORI-934 in 2010 near the submarine mud volcano MV4 located at the upper slope of south-west Taiwan. The methanogenic substrates utilized by strain S3Fa^Twere formate and H₂/CO₂but not acetate, secondary alcohols, methylamines, methanol or ethanol. Cells of strain S3Fa^Twere non-motile, irregular cocci, 0.5–1.0 μm in diameter. The surface-layer protein showed an Mr of 128 000.The optimum growth conditions were 37 °C, pH 7.1 and 0.17 M NaCl. The DNA G+C content of the genome of strain S3Fa^Twas 62.3 mol %. Phylogenetic analysis revealed that strain S3Fa^Twas most closely related toMethanoculleus marisnigriJR1^T(99.3 % 16S rRNA gene sequence similarity). Genome relatedness between strain S3Fa^TandMethanoculleus marisnigriJR1^Twas computed using both genome-to-genome distance analysis (GGDA) and average nucleotide identity (ANI) with values of 46.3–55.5 % and 93.08 %, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data, it is evident that strain S3Fa^Trepresents a novel species of the genusMethanoculleus, for which the nameMethanoculleus sediminissp. nov. is proposed. The type strain is S3Fa^T( = BCRC AR10044^T = DSM 29354^T).

Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan. The cells of strain P2F9704aT were non-motile, irregular cocci 0.9-1.4 microm in diameter. They stained gram-negative. The cells catabolized formate and H2+CO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates. The optimal growth parameters for strain P2F9704aT were pH 6.7, 37 degrees C and 0.5% NaCl. Acetate was required for cell growth even though it was not a substrate for methanogenesis. The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT. However, tungsten extended the growth ranges relating to temperature, pH and salt. The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 99.1 and 98.5% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species. On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis. The type strain is P2F9704aT (= OCM 671T = CCRC 16182T = DSM 14663T).

A mesophilic, hydrogenotrophic methanogen, strain CYW4^T, was isolated from deep-sea sediment obtained by the Ocean Researcher I cruiser, ORI-961, in 2011. The sediment was from the deformation front area offshore of south-western Taiwan. Here, seismic reflections indicated that methane hydrates were abundant. The methanogenic substrates utilized by strain CYW4^Twere formate and H₂/CO₂, but not acetate, secondary alcohols, methylamines, methanol and ethanol. Cells of strain CYW4^Twere non-motile, irregular cocci and 0.6–1.5 µm in diameter. The S-layer protein had anM_rof 112 000. The optimum growth conditions were at 37 °C, pH 8.1 and 0.08 M NaCl. Growth of the strain was stimulated by acetate. The G+C content of the chromosomal DNA of strain CYW4^Twas 61 mol%. Phylogenetic analysis revealed that strain CYW4^Twas most closely related to Methanoculleus marisnigri JR1^T(96.82 % 16S rRNA gene sequence similarity). Based on the morphological, phenotypic and phylogenetic characteristics presented here, it is evident that strain CYW4^Trepresents a novel species of the genus Methanoculleus , and the nameMethanoculleus taiwanensissp. nov. is proposed. The type strain is CYW4^T( = BCRC AR10043^T = NBRC 110782^T). The optical density of cultures of strain CYW4^Tdropped abruptly upon entering the stationary growth phase. During this time numerous particles of approximately 50 nm in diameter were observed on and around the cells. This suggests that strain CYW4^Tharbours a lytic virus that is induced in the stationary phase, which is of interest because only a few lytic viruses have been reported in methanogens.

A mesophilic, hydrogenotrophic methanogen, strain ML15^T, was isolated from an aquaculture fish pond near Wang-gong, Taiwan. The cells were irregular cocci, non-motile, 1·5–2·0 μm in diameter and Gram-negative. Cells of strain ML15^Tlysed easily in the presence of SDS (0·1 g l⁻¹) and the S-layer protein had anM_rof 138 800. The catabolic substrates utilized by this strain included formate and H₂/CO₂, but not acetate, methanol, trimethylamine or secondary alcohols. Growth did not occur in minimal medium, but was observed when yeast extract and tryptone were added. Strain ML15^Tgrew fastest at 37 °C, pH 6·6–7·0 and with 3 % NaCl. Acetate was not required for cell growth. Trace amounts of tungstate promoted cell growth. The G+C contents of DNA ofMethanofollis aquaemarisN2F9704^Tand strain ML15^Twere 59·1 and 58·4 mol%, respectively. Sequence analysis of the 16S rRNA genes of strain ML15^Tand selectedMethanofollisspecies revealed similarities of 95–97 %. Based on the data presented here, it is proposed that strain ML15^T(=OCM 789^T=DSM 15483^T) represents a novel species,Methanofollis formosanussp. nov.

This study reports a new white-rot fungus Cerrena sp. WR1, identified based on an 18S rDNA sequence, which can secrete extracellular forms of laccase with a maximal activity reaching 202 000 U l⁻¹ in a 5-l fermenter. A laccase protein, designated Lcc3, was purified and shown to be N-linked glycosylated by PNGase F and liquid chromatography tandem mass spectrometry analyses. The respective full-length cDNA gene (lcc3) of the Lcc3 protein was obtained using polymerase chain reaction-based methods. Kinetic studies showed that the K_m and k_cat of the native Lcc3 were 3.27 μM and 934.6 s⁻¹ for 2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid), 849.1 μM and 147.9 s⁻¹ for guaiacol, 392.7 μM and 109.2 s⁻¹ for 2,6-dimethoxyphenol, and 881 μM and 115.5 s⁻¹ for catechol, respectively. The T_m of Lcc3 was determined at 73.9°C and it showed a long t_1/2 (120 min) at 50°C. The laccase was highly ethanol resistant, with 80% of its original activity was detected when incubated in 25% ethanol for 14 days. Furthermore, crude enzyme broth or Lcc3 could degrade lignin in kraft paper (26.5%), and showed high decoloration efficiency (90%) on synthetic dye Remazol Brilliant Blue R. Together, these data demonstrate that Cerrena sp. WR1 Lcc3 possesses novel biochemical and kinetic properties that may aid its application in industry.