Growth curves and methane synthesis of <i>M. acetivorans</i> cultured on methanol (A, C) or acetate (B, D), respectively, and in the absence (filled squares) or presence of 100 µM CdCl₂ (open squares).

<p>Values represent the mean ± SD of at least 4 different cell batches. ^a: P<0.05 <i>vs</i> control curve without cadmium using two way ANOVA. Inset; curves with 1 (filled circles), 10 (filled triangles), 25 (open squares) and 50 (open circles) µM CdCl₂.</p

Activation of methane synthesis by cadmium.

<p>(A) 1, 10 and 100 µM of CdCl₂ were added and methane production was determined after 5 min in acetate-grown control cells. (B) Short-term methane synthesis in the absence (open symbols) or presence (filled symbols) of 10 µM CdCl₂ in methanol- (▪) and acetate-grown cells (•). These experiments were started after thoroughly purging the flasks and adding the indicated CdCl₂ concentrations (time-point equal to zero). (C) Activation of methane synthesis by other heavy metals. Acetate-grown cells cultures were incubated for 5 min in the absence or presence of 100 µM of the metals indicated. At t = 0 (before metal addition), the methane remaining in the bottle cultures was 8.8±1.2 µmol methane <i>per</i> culture. P<0.05 using the Student's t-test for non-paired samples for ^a<i>vs</i> control (without cadmium or other metal ion); ^b<i>vs</i> cells exposed to 1 µM cadmium; ^c<i>vs</i> methanol cultures exposed to cadmium.</p

Activation of Methanogenesis by Cadmium in the Marine Archaeon <em>Methanosarcina acetivorans</em>

<div><p><em>Methanosarcina acetivorans</em> was cultured in the presence of CdCl₂ to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd²⁺ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.</p> </div

Methane production and cadmium accumulation in <i>M. acetivorans</i> cultured on acetate or methanol.

<p>Data shown were obtained from cell cultures at the end of the growth curve. Values are the mean ± SD of at least 4 cultures from different batches.</p>a<p>: P<0.05 <i>vs</i> acetate-grown cells at any other concentration of cadmium;</p>b<p>: P<0.05 <i>vs</i> methanol-grown cells at any other concentration of cadmium;</p>c<p>acetate-grown cells <i>vs</i> 25, 50 and 100 µM cadmium, using the Student's t-test.</p

Effect of cadmium on enzyme activities of the acetoclastic pathway upper part from <i>Methanosarcina acetivorans</i>.

<p>All activities were determined by using freshly prepared cytosolic fraction as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048779#s2" target="_blank">Methods</a> section. Values are the mean ± SD of at least three independent preparations.</p><p>Control activities were for AK: 0.75±0.21 U/mg protein (n = 4); for Pta: 1.48±0.8 U/mg protein (n = 4); for CODH/AcCoA synthase with acetyl-CoA: 0.37 U±0.12 U/mg protein (n = 5); and with CO: 0.68±0.11 U/mg protein (n = 3); CA: 26±12 U/mg protein.</p>*<p>P<0.05 <i>vs</i> control for independent samples;</p>**<p>P<0.05 <i>vs</i> control for paired samples. ND: Not determined.</p

Air-Adapted <i>Methanosarcina acetivorans</i> Shows High Methane Production and Develops Resistance against Oxygen Stress

<div><p><i>Methanosarcina acetivorans</i>, considered a strict anaerobic archaeon, was cultured in the presence of 0.4–1% O₂ (atmospheric) for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O₂ were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells). In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O₂. Production of reactive oxygen species was 50 times lower in air-adapted cells <i>versus</i> control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O₂ toxicity. In this regard, (i) the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii) the thiol-molecules (cysteine + coenzyme M-SH + sulfide) and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells <i>versus</i> anaerobic-control cells. Long-term cultures (18 days) of air-adapted cells exposed to 2% O₂ exhibited the ability to form biofilms. These data indicate that <i>M. acetivorans</i> develops multiple mechanisms to contend with O₂ and the associated oxidative stress, as also suggested by genome analyses for some methanogens.</p></div

Formation of a biofilm extracellular matrix induced by O₂ stress in <i>M</i>. <i>acetivorans</i>.

<p>Samples were prepared for and analyzed by scanning electron microscope as described in the Methods section. Extracellular matrix was absent in cells grown in the absence of oxygen (A) but well-defined in air-adapted cells (B). The carbon source was methanol, which was initially 100 mM and further replenished every 3 days. Micrographs are shown at 5000X, bar = 2 μm. (C) Biofilm formation determined by crystal violet staining in control-grown cells and air adapted cells cultured in methanol. *<i>P</i> < 0.01 <i>versus</i> control cells. (D) Extracellular DNA determination; 1 mg protein of control (filled squares) and air adapted cells (open circles) was added to a quartz cuvette with 1.8 mL of TME buffer. After 60 seconds of baseline acquisition, DNAse I was added and the light pass was determined at 540 nm.</p

Methane synthesis and oxygen consumption in <i>M</i>. <i>acetivorans</i>.

<p>Cells were cultured in methanol (A, C) or acetate (B, D) and the contents of methane (A, B) and O₂ (C, D) in the head space at the indicated times were determined. Control anaerobic cells (■), control anaerobic cells <i>plus</i> air pulses (●) and air-adapted cells (▲). The O₂ concentration in the head space of culture bottles without cells was also determined (□). The increase in the content of O₂ was due to each new air injection which was indicated by arrows. Values are the mean ± SD of at least 4 different independent cell batches. ^a P<0.05; ^b P <0.01 <i>vs</i> air-adapted cells.</p

Transcript levels of anti-oxidant genes in <i>M</i>. <i>acetivorans</i>.

<p>Anaerobic control cells grown on acetate and harvested in the stationary phase were incubated at 37°C under orbital shaking in the absence (lane 1) or presence (lane 2) of 2% O₂ for 2 h. Air-adapted cells grown and harvested in the same conditions were also exposed to O₂ (lane 3). mRNA isolation and RT-PCR analysis was carried out by the primer dropping method as indicated in the Methods section and the PCR products separated by gel electrophoresis (A). Densitometric analysis (B) was carried out by double normalization <i>versus</i> the internal control MA3998 transcript and the target genes from anaerobic control cultures without O₂ exposure (lane 1). Values are the mean of 4 independent experiments ± SD.</p

Antioxidant enzyme activities.

<p>Cytosolic-enriched fractions from anaerobic control (black bars), anaerobic <i>plus</i> 2% O₂ for 2h (grey bars) and air-adapted cells (white bars) grown on methanol (A) or acetate (B) were used to determine activities of SOD, APX and CAT as described under Methods. Rate units for APX are mU (nmol of substrate consumed (min x mg protein)^-1), whereas CAT and SOD activity units are U (mg protein)^-1 as described under Methods. Values shown are the mean ± SD of at least 4 independent preparations. *P<0.01; ** P<0.05 <i>vs</i> anaerobic control cells. ^a P<0.01 <i>vs</i> methanol-grown cells.</p