In a sealed 10 mL bottle with rubber stopper. To detect the CO2 formation, 5 mL of the head space was taken and injected at different times (0, 30, 60 and 120 s) in a gas chromatograph equipped with a Thermal Conductivity Detector. CO2 formed in assay reaction buffer without enzyme and with boiled enzyme was subtracted from the CO2 formed with enzyme (representative traces are shown in figures S3 and S4).of cadmium removed 240 h(acetate) or 96 h (methanol)Methanol38623 5569 9406326b 13876225 4.160.03 4.660.2 9.663.8 5.960.6acetate1664cCd removed and accumulated nmol/total cell protein862c10.8.364.Methane produced mmol/240 h (acetate) or 96 h (methanol)methanol4.0860.4.160.4.160.4.560.acetate4.560.4.360.methanol4.460.4.660.8.961.mg of total protein/culture5.261.acetate9.663.10.7.961.5.660.Estimated Free [Cd2+] pM5.860.21.2.Total [CdCl2] mM6.460.5.161.8.962.4.760.4.160.Acetatea20546929bmethanolBiogas Production and Metal Removal(DTNB) and spectrophotometrically (methylene blue), respectively (mean 6 SE, n = 4); the ionic 301-00-8 strength = 0.77, pH = 7.0 and temperature = 36uC. The log values of the equilibrium constants (Keq) for the association of the complexes were 13.4 and 20.13 for Cys-cadmium and Cys-Cd-Cys, and 6.1 for sulfide-cadmium [20]. Cells cultured on methanol showed similar growth either in the absence or presence of up to 100 mM total CdCl2 (Fig. 1A and inset). With acetate, growth was slightly faster in cultures with 100 mM cadmium during the exponential phase (Fig. 1B and inset). To undoubtedly establish that the turbidity increase induced by cadmium was indeed reporting cell growth in acetate cultures, the growth rate (GR) and the duplication time (DT) were determined by using the curve of methane production vs time and assuming that methane production is proportional to the number of living cells present in the culture. The duplication times were similar to those reported previously for M. acetivorans [11]. No significant differences in GR values (0.06460.003 versus 0.062560.003 h21) and DT values (1062.3 h versus 1162.7 h) were found for cells cultured in methanol without or with 100 mM total Cd2+. In contrast, in acetate cultures the GR value was BIBS39 price significantly higher in cultures with 100 mM Cd2+ (0.02860.004 versus 0.03060.006 h21; n = 5, P,0.05). DT did not significantly changed (2663 versus 2462 h; n = 5) for cells cultured without or with 100 mM Cd, respectively (Fig. 1). Furthermore, two way ANOVA analyses on the global data showed that cadmium exerted a positive effect on the growth curve with acetate but not with methanol as carbon source (Figs. 1C and 1D). The rate of methane production in acetate cultures with 100 mM total CdCl2 was slightly but significantly higher than in itsabsence in the time-period from 110 up to 230 h of culture (Fig. 1D). It is worth noting that the methane yield, at the end of the growth curves (96 and 244 h for methanol and acetate, respectively), was the same under all conditions, because the total amount of carbon source added was identical (Table 1): 4.160.13 (control) and 4.160.02 mmol methane (+100 mM CdCl2) for methanol and 4.560.3 (control) and 4.760.3 mmol methane (+100 mM CdCl2) for acetate. On the other hand, when 500 mM total CdCl2 was added to cultures with acetate or methanol as carbon source, no growth or methane synthesis were detected (data not shown) indicating that these high cadmium levels were indeed extremely toxic to the cells.Effect of cadmium on methane synthesisFor s.In a sealed 10 mL bottle with rubber stopper. To detect the CO2 formation, 5 mL of the head space was taken and injected at different times (0, 30, 60 and 120 s) in a gas chromatograph equipped with a Thermal Conductivity Detector. CO2 formed in assay reaction buffer without enzyme and with boiled enzyme was subtracted from the CO2 formed with enzyme (representative traces are shown in figures S3 and S4).of cadmium removed 240 h(acetate) or 96 h (methanol)Methanol38623 5569 9406326b 13876225 4.160.03 4.660.2 9.663.8 5.960.6acetate1664cCd removed and accumulated nmol/total cell protein862c10.8.364.Methane produced mmol/240 h (acetate) or 96 h (methanol)methanol4.0860.4.160.4.160.4.560.acetate4.560.4.360.methanol4.460.4.660.8.961.mg of total protein/culture5.261.acetate9.663.10.7.961.5.660.Estimated Free [Cd2+] pM5.860.21.2.Total [CdCl2] mM6.460.5.161.8.962.4.760.4.160.Acetatea20546929bmethanolBiogas Production and Metal Removal(DTNB) and spectrophotometrically (methylene blue), respectively (mean 6 SE, n = 4); the ionic strength = 0.77, pH = 7.0 and temperature = 36uC. The log values of the equilibrium constants (Keq) for the association of the complexes were 13.4 and 20.13 for Cys-cadmium and Cys-Cd-Cys, and 6.1 for sulfide-cadmium [20]. Cells cultured on methanol showed similar growth either in the absence or presence of up to 100 mM total CdCl2 (Fig. 1A and inset). With acetate, growth was slightly faster in cultures with 100 mM cadmium during the exponential phase (Fig. 1B and inset). To undoubtedly establish that the turbidity increase induced by cadmium was indeed reporting cell growth in acetate cultures, the growth rate (GR) and the duplication time (DT) were determined by using the curve of methane production vs time and assuming that methane production is proportional to the number of living cells present in the culture. The duplication times were similar to those reported previously for M. acetivorans [11]. No significant differences in GR values (0.06460.003 versus 0.062560.003 h21) and DT values (1062.3 h versus 1162.7 h) were found for cells cultured in methanol without or with 100 mM total Cd2+. In contrast, in acetate cultures the GR value was significantly higher in cultures with 100 mM Cd2+ (0.02860.004 versus 0.03060.006 h21; n = 5, P,0.05). DT did not significantly changed (2663 versus 2462 h; n = 5) for cells cultured without or with 100 mM Cd, respectively (Fig. 1). Furthermore, two way ANOVA analyses on the global data showed that cadmium exerted a positive effect on the growth curve with acetate but not with methanol as carbon source (Figs. 1C and 1D). The rate of methane production in acetate cultures with 100 mM total CdCl2 was slightly but significantly higher than in itsabsence in the time-period from 110 up to 230 h of culture (Fig. 1D). It is worth noting that the methane yield, at the end of the growth curves (96 and 244 h for methanol and acetate, respectively), was the same under all conditions, because the total amount of carbon source added was identical (Table 1): 4.160.13 (control) and 4.160.02 mmol methane (+100 mM CdCl2) for methanol and 4.560.3 (control) and 4.760.3 mmol methane (+100 mM CdCl2) for acetate. On the other hand, when 500 mM total CdCl2 was added to cultures with acetate or methanol as carbon source, no growth or methane synthesis were detected (data not shown) indicating that these high cadmium levels were indeed extremely toxic to the cells.Effect of cadmium on methane synthesisFor s.
