Comparative characteristics of strain HZ254^T and <i>Methanocella paludicola</i> SANAE^T and <i>Methanocella arvoryzae</i> MRE50^T.

<p>Data for strain HZ254^T is from this study, and strain SANAE^T and MRE50^T were retrieved from Sakai <i>et al.</i>, 2008 and 2010.</p>*<p>The data in parentheses were determined by HPLC, other data were taken from genome information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Erkel1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-L1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Sakai4" target="_blank">[24]</a>.</p>†<p>pH for HZ254^T and other strains were determined at 55°C and 25°C, respectively. Abbreviations, −, negative; +, positive; N.A., not applicable.</p

T-RFLP patterns based on 16S rRNA genes for enrichment cultures of strain HZ254^T along with successive transfers.

<p>The analysis was performed using Ar109f/915r primer set and <i>TaqI</i> restriction enzymes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Peng1" target="_blank">[14]</a>. T-RFLP fingerprints were normalized to a total of 100 relative fluorescence units (RFU), and T-RF peaks with RFU less than 1 were discarded. The 254-bp T-RF was affiliated with <i>Methanocellales</i> (RC-I) as determined by cloning and sequencing of 16S rRNA genes, and the T-RF length calculated from the sequence was actually 258-bp (data not shown). All other T-RF peaks could be assigned correspondingly to <i>Methanomicrobiales</i> (Mm), <i>Methanobacteriales</i> (Mb), <i>Methanosarcinaceae</i> (Msr)/Crenarchaeotal group 1.1b (G1.1b), <i>Methanosaetaceae</i> (Msa) and RC-I/<i>Methanomicrobiales</i> (Mm), according to our previous studies in the same soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Peng1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Yuan1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Wu1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Yuan2" target="_blank">[36]</a>, respectively. The pre-incubation samples were sampled after 24 hours of incubation, and all other samples were sampled after that methane production ceased and/or hydrogen could not be detected in the headspace. After the 13^th transfer, the archaeal community was still frequently monitored by T-RFLP analysis along with subsequent transfers, but the 254-bp was always the sole T-RF product.</p