Opper inhibitory effect was previously described for methanogenesis derived from anaerobic sludge digestion [10,21]. On this regard, a positive effect of pM concentrations cadmium on growth presumably due to activation of CA was reported for Thalassiosira weissflogii, a diatom that also comes from the marine habitat [22]. Interestingly, acetate-grown M. acetivorans cells have significant higher AK, Pta, CODH/AcCoAs and CA protein content than methanol-grown cells [23,24]. Hence, the influence of cadmium on enzymes activities from the upper part of theBiogas Production and Metal RemovalTable 2. Effect of cadmium on enzyme activities of the acetoclastic pathway upper part from Methanosarcina acetivorans.[CdCl2] mMAcetate kinase timesPhosphotranacetylase timesCODH/AcCoA synthase times with LY2409021 web acetyl-CoA with CO 1 1.0360.11 0.6660.21 0.6060.16 0.560.16 NDCarbonic anhydrase times0 0.01 0.1 1 101 1.3860.27 1.3560.18** 1.160.16 Not determined ND1 Not determined Not determined 0.7360.15 0.7760.17 0.4760.1 1.060.1 1.0260.3 0.9860.18 0.7460.2 ND1 2.960.8 ** 4.963.2 * 4.262.7 * 1.760.8 NDAll activities were determined by using freshly prepared cytosolic fraction as described in the 79831-76-8 site Methods section. Values are the mean 6 SD of at least three independent preparations. Control activities were for AK: 0.7560.21 U/mg protein (n = 4); for Pta: 1.4860.8 U/mg protein (n = 4); for CODH/AcCoA synthase with acetyl-CoA: 0.37 U60.12 U/mg protein (n = 5); and with CO: 0.6860.11 U/mg protein (n = 3); CA: 26612 U/mg protein. *P,0.05 vs control for independent samples; **P,0.05 vs control for paired samples. ND: Not determined. doi:10.1371/journal.pone.0048779.tFigure 3. Identification of cadmium clusters in M. acetivorans. Spectral analysis by HAADF-STEM from methanol-grown cells with 100 mM CdCl2 (A) or without cadmium (B). C: carbon; O: oxygen, Cu: cupper; S: sulfur; Cd: cadmium. doi:10.1371/journal.pone.0048779.gBiogas Production and Metal Removalreported [18] for HAADF-STEM. Although the images were diffused (Fig. S6), high intracellular contents of electro-dense grains of cadmium and sulfur were definitively identified (Fig. 3). A similar accumulation profile has been reported for cobalt, where the complexes cobalt-chloride and cobalt-citrate are the forms that preferentially are retained in the granular sludge and affect methane production [33]. Such high capacity for cadmium removal in M. acetivorans suggests that this Archaea may have developed strategies to contend against heavy metals different to those reported for the eubacteria domain, which are mainly based on the extrusion of the metal by means of pumps that use ATP as motive force [34]. It has been documented that an excess of zinc, copper, or cobalt, all essential heavy metals, inhibit methanogenesis [9,31,35,36]. In contrast, cadmium seems to be less toxic for methanogenesis in the granular sludge [10], although the free metal concentration or the organic complexes formed were not described. In the present study the free Cd2+ concentration in the culture medium was estimated to be in the pM range. This suggested that M. acetivorans has very high affinity transporters for Cd2+ or that the metal ion may permeate the plasma membrane as a complex with the external cysteine and sulfur.Figure S3 Formation of CO2 by CA from M. acetivorans in the absence (chromatogram A), or presence of 1 mM CdCl2. In chromatogram C, cytosolic fraction was previously heated. The different peaks shown represent consecutive.Opper inhibitory effect was previously described for methanogenesis derived from anaerobic sludge digestion [10,21]. On this regard, a positive effect of pM concentrations cadmium on growth presumably due to activation of CA was reported for Thalassiosira weissflogii, a diatom that also comes from the marine habitat [22]. Interestingly, acetate-grown M. acetivorans cells have significant higher AK, Pta, CODH/AcCoAs and CA protein content than methanol-grown cells [23,24]. Hence, the influence of cadmium on enzymes activities from the upper part of theBiogas Production and Metal RemovalTable 2. Effect of cadmium on enzyme activities of the acetoclastic pathway upper part from Methanosarcina acetivorans.[CdCl2] mMAcetate kinase timesPhosphotranacetylase timesCODH/AcCoA synthase times with Acetyl-CoA with CO 1 1.0360.11 0.6660.21 0.6060.16 0.560.16 NDCarbonic anhydrase times0 0.01 0.1 1 101 1.3860.27 1.3560.18** 1.160.16 Not determined ND1 Not determined Not determined 0.7360.15 0.7760.17 0.4760.1 1.060.1 1.0260.3 0.9860.18 0.7460.2 ND1 2.960.8 ** 4.963.2 * 4.262.7 * 1.760.8 NDAll activities were determined by using freshly prepared cytosolic fraction as described in the Methods section. Values are the mean 6 SD of at least three independent preparations. Control activities were for AK: 0.7560.21 U/mg protein (n = 4); for Pta: 1.4860.8 U/mg protein (n = 4); for CODH/AcCoA synthase with acetyl-CoA: 0.37 U60.12 U/mg protein (n = 5); and with CO: 0.6860.11 U/mg protein (n = 3); CA: 26612 U/mg protein. *P,0.05 vs control for independent samples; **P,0.05 vs control for paired samples. ND: Not determined. doi:10.1371/journal.pone.0048779.tFigure 3. Identification of cadmium clusters in M. acetivorans. Spectral analysis by HAADF-STEM from methanol-grown cells with 100 mM CdCl2 (A) or without cadmium (B). C: carbon; O: oxygen, Cu: cupper; S: sulfur; Cd: cadmium. doi:10.1371/journal.pone.0048779.gBiogas Production and Metal Removalreported [18] for HAADF-STEM. Although the images were diffused (Fig. S6), high intracellular contents of electro-dense grains of cadmium and sulfur were definitively identified (Fig. 3). A similar accumulation profile has been reported for cobalt, where the complexes cobalt-chloride and cobalt-citrate are the forms that preferentially are retained in the granular sludge and affect methane production [33]. Such high capacity for cadmium removal in M. acetivorans suggests that this Archaea may have developed strategies to contend against heavy metals different to those reported for the eubacteria domain, which are mainly based on the extrusion of the metal by means of pumps that use ATP as motive force [34]. It has been documented that an excess of zinc, copper, or cobalt, all essential heavy metals, inhibit methanogenesis [9,31,35,36]. In contrast, cadmium seems to be less toxic for methanogenesis in the granular sludge [10], although the free metal concentration or the organic complexes formed were not described. In the present study the free Cd2+ concentration in the culture medium was estimated to be in the pM range. This suggested that M. acetivorans has very high affinity transporters for Cd2+ or that the metal ion may permeate the plasma membrane as a complex with the external cysteine and sulfur.Figure S3 Formation of CO2 by CA from M. acetivorans in the absence (chromatogram A), or presence of 1 mM CdCl2. In chromatogram C, cytosolic fraction was previously heated. The different peaks shown represent consecutive.
