MOESM1 of Changes in the microbial consortium during dark hydrogen fermentation in a bioelectrochemical system increases methane production during a two-stage process

Additional file 1: Fig. S1. Reactor performances of the two-stage processes of the bioelectrochemical system (BES)→methane fermenter (MF) and non-bioelectrochemical system (NBES) →methane fermenter (MF). (a) Rate of gas production of hydrogen, methane, and carbon dioxide. (b) Production of soluble metabolic products. (c) Carbon balance of the system. The organic loading rate (OLR) of the BES and NBES was 2207.6 mg-C/L/day, corresponding to a glucose load of 27.8 mMglucose/L/day. The pH of the substrate was 7.3. The hydraulic retention time (HRT) of the BES and NBES was 2 days and the HRT of second-stage MFs was 4 days

Nicotine transport activity of Nt-JAT2.

<p>(A) Time course analysis of nicotine transport in Nt-JAT2-expressing yeast. Control (dashed line) and Nt-JAT2-expressing (solid line) yeast cells were incubated in half-strength SD medium supplemented with 1 mM nicotine and sampled at the times indicated. Results are mean ± SD of triplicates. *<i>P</i><0.05; **<i>P</i><0.01 compared with control by Student’s t-test. (B) Localization of Nt-JAT2–GFP in yeast cells. Yeast cells expressing Nt-JAT2–GFP were grown at 30°C to logarithmic growth phase and observed by fluorescence microscopy. (i) Fluorescence of yeast cells transformed with Nt-JAT2–GFP; (ii) bright-field contrast (scale bar, 5 µm). (C) Nicotine content in control (white bar), Nt-JAT1-expressing (gray bar) and Nt-JAT2 -expressing (black bar) yeast cells incubated in half-strength SD medium containing 0.5 mM nicotine for 6 h. Results are mean ± SD of triplicates. *<i>P</i><0.01 compared with control by Student’s t-test.</p

MeJA induction of <i>Nt-JAT2</i> mRNA expression in tobacco seedlings.

<p>(A) <i>Nt-JAT2</i> expression in response to various treatments. Fourteen-day-old seedlings were treated for 24 h with 10 µM 1-naphthaleneacetic acid (NAA), 10 µM IAA, 10 µM 6-benzyladenine (BA), 10 µM abscisic acid (ABA), 10 µM gibberellic acid (GA₃), 5 µM brassinolide (BL), 100 µM MeJA, 100 µM salicylic acid (SA), or 100 µM sclareol (SC), at 4°C (cold)/low light, dark (dark), and drought (dry) conditions. Cont., untreated control. Total RNA (7.5 µg) prepared from the aerial parts of seedlings was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (top). Loading controls are shown as EtBr-stained 18S rRNA (bottom). (B) RNA gel blot analysis of <i>Nt-JAT2</i> in tobacco seedlings. Seedlings were harvested 0 to 72 h after MeJA treatment. Total RNA (7.5 µg) was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). Loading control is shown as EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2, expression analyses were performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>.</p

Expression of Nt-JAT2 in tobacco plants.

<p>(A) Organ-specific expression of <i>Nt-JAT2</i> mRNA in tobacco plants. Total RNA (7.5 µg) prepared from each tobacco organ was probed with ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). The amount of total RNA applied to each lane is shown by EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2 expression, analysis was performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>. (B, C) Immunoblot analysis of Nt-JAT2 and Nt-JAT1 proteins in control (B) and MeJA treated (C) plants. Microsomes from tobacco leaves, stems and roots were subjected to electrophoresis, blotted, and incubated with antibody to Nt-JAT2 or Nt-JAT1. L, leaves (Leaves were numbered from top to bottom).</p

Subcellular localization of Nt-JAT2 in cultured tobacco cells.

<p>(A) Confocal images of Nt-JAT2-GFP in transgenic BY-2 cells. (left) Nt-JAT2-GFP fluorescence images, (right) bright field images, (bottom left) merged images. (B) Nt-JAT2-GFP fluorescence (green, top left), FM4-64 fluorescence (red, bottom left), merged (bottom right), and bright field (top right) images after treatment for 24 h (scale bar, 2 µm.).</p