Hort-term experiments, cultures of cells in the early stationary growth phase (10 days for acetate- and 4 days for methanol-grown cells) were incubated with CdCl2 at 25?7uC. The concentrations of acetate and methanol remaining in the cultures were 863 mM (4006150 mmol acetate; n = 5) and 561 mM (250650 mmol methanol; n = 5), respectively. Under these conditions, cadmium exerted a remarkably stimulating effect on the synthesis of methane in control cells not previously exposed to Cd2+; the most potent activation was reached at 10 mM total CdCl2 (Fig. 2A). Moreover, the rate of the methane production increased 9 and 6.5 fold for acetate- and methanol-grown cells, respectively, in 2 min (Fig. 2B). After 10 min of incubation the methane produced, in the absence or presence of 10 mM total CdCl2, by stationary acetategrown cells was 1863 and 2664 mmol methane, and after 60 minFigure 1. Growth curves and methane synthesis of M. acetivorans cultured on methanol (A, C) or acetate (B, D), respectively, and in the absence (filled squares) or presence of 100 mM CdCl2 (open squares). Values represent the mean 6 SD of at least 4 different cell batches. a: P,0.05 25331948 vs control curve without cadmium using two way ANOVA. Inset; curves with 1 (filled circles), 10 (filled triangles), 25 (open squares) and 50 (open circles) mM CdCl2. doi:10.1371/journal.pone.0048779.gBiogas Production and Metal RemovalFigure 2. Activation of methane synthesis by cadmium. (A) 1, 10 and 100 mM 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 mM 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. Acetategrown cells cultures were incubated for 5 min in the absence or presence of 100 mM of the metals indicated. At t = 0 (before metal addition), the methane remaining in the bottle cultures was 8.861.2 mmol methane per culture. P,0.05 using the Student’s t-test for non-paired samples for a vs control (without cadmium or other metal ion); b vs cells exposed to 1 mM cadmium; c vs methanol cultures exposed to cadmium. doi:10.1371/journal.pone.0048779.gNaceticlastic pathway, which have not been previously determined in M. acetivorans, was here examined (Table 2). AK activity was 10 fold lower (see legend to Table 2 for values) than that reported for the enzyme from M. thermophila [25]; the AK activity slightly increased (25?0 ) by 10 mM total cadmium. This cadmium activating effect is intriguing because no metal has been reported to be required for AK activity in M. thermophila [26]. Pta activity under our conditions was 15 times lower than that reported for the enzyme from M. thermophila [27], whereas the CODH/AcCoAs activity determined in the present work was 10 times higher than that reported for the enzyme from M. thermophila [17]. The last two enzymes were not activated by 0.01?0 mM total CdCl2, but they were rather partially inhibited (Table 2). With a novel strategy to determine CA activity which was based on Sapanisertib measuring by gas HC-030031 chromatography the CO2 produced, the M. acetivorans CA showed a higher activity than that reported by semiquantitative electrometric method at alkaline pH for the M. thermophila enzyme [28] and marked activation b.Hort-term experiments, cultures of cells in the early stationary growth phase (10 days for acetate- and 4 days for methanol-grown cells) were incubated with CdCl2 at 25?7uC. The concentrations of acetate and methanol remaining in the cultures were 863 mM (4006150 mmol acetate; n = 5) and 561 mM (250650 mmol methanol; n = 5), respectively. Under these conditions, cadmium exerted a remarkably stimulating effect on the synthesis of methane in control cells not previously exposed to Cd2+; the most potent activation was reached at 10 mM total CdCl2 (Fig. 2A). Moreover, the rate of the methane production increased 9 and 6.5 fold for acetate- and methanol-grown cells, respectively, in 2 min (Fig. 2B). After 10 min of incubation the methane produced, in the absence or presence of 10 mM total CdCl2, by stationary acetategrown cells was 1863 and 2664 mmol methane, and after 60 minFigure 1. Growth curves and methane synthesis of M. acetivorans cultured on methanol (A, C) or acetate (B, D), respectively, and in the absence (filled squares) or presence of 100 mM CdCl2 (open squares). Values represent the mean 6 SD of at least 4 different cell batches. a: P,0.05 25331948 vs control curve without cadmium using two way ANOVA. Inset; curves with 1 (filled circles), 10 (filled triangles), 25 (open squares) and 50 (open circles) mM CdCl2. doi:10.1371/journal.pone.0048779.gBiogas Production and Metal RemovalFigure 2. Activation of methane synthesis by cadmium. (A) 1, 10 and 100 mM 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 mM 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. Acetategrown cells cultures were incubated for 5 min in the absence or presence of 100 mM of the metals indicated. At t = 0 (before metal addition), the methane remaining in the bottle cultures was 8.861.2 mmol methane per culture. P,0.05 using the Student’s t-test for non-paired samples for a vs control (without cadmium or other metal ion); b vs cells exposed to 1 mM cadmium; c vs methanol cultures exposed to cadmium. doi:10.1371/journal.pone.0048779.gNaceticlastic pathway, which have not been previously determined in M. acetivorans, was here examined (Table 2). AK activity was 10 fold lower (see legend to Table 2 for values) than that reported for the enzyme from M. thermophila [25]; the AK activity slightly increased (25?0 ) by 10 mM total cadmium. This cadmium activating effect is intriguing because no metal has been reported to be required for AK activity in M. thermophila [26]. Pta activity under our conditions was 15 times lower than that reported for the enzyme from M. thermophila [27], whereas the CODH/AcCoAs activity determined in the present work was 10 times higher than that reported for the enzyme from M. thermophila [17]. The last two enzymes were not activated by 0.01?0 mM total CdCl2, but they were rather partially inhibited (Table 2). With a novel strategy to determine CA activity which was based on measuring by gas chromatography the CO2 produced, the M. acetivorans CA showed a higher activity than that reported by semiquantitative electrometric method at alkaline pH for the M. thermophila enzyme [28] and marked activation b.
