Cultures were grown in RM medium (10 g/L yeast extract, 2 g/L KH2PO4) supplemented with either 2% (w/v) glucose (RMG) or 2% (w/v) xylose (RMX) for inhibitor studies. The pH of all media was adjusted to pH5.8 and was filter sterilized. Media were prepared from stock solutions of yeast extract and monobasic potassium phosphate, and when possible, inhibitor stock solutions were prepared and titrated to pH 5.8. Two compounds (4-hydroxycinnamic acid and syringaldehyde) were prepared from stock solutions in 100% DMSO due to their low solubility in water. The total amount of DMSO in the final medium ranged from 0.1-5.0% (v/v) for 4-hydroxycinnamic acid and 0.1%-3% (v/v) in syringaldehyde. Inhibitor studies with DMSO alone did not detect notable inhibitions on growth or final cell mass (data not shown).
Ammonium salts were prepared by titrating the acids with concentrated ammonium hydroxide, except for the anions: sulfate, chloride, nitrate, acetate, phosphate. Calcium formate was prepared by titrating formic acid with lime. Phosphate stocks were made by preparing 1 M stock solutions of monobasic phosphate and titrating the medium to pH of 5.8 with 1 M dibasic solution.
As a consequence of precipitation of monobasic potassium phosphate with calcium, it was not included in medium containing calcium. In this case, 50 mM MES, pH 5.8, was provided to supply some buffering capacity. Growth rates in RMG or RMX with 50 mM MES, pH 5.8, and no potassium phosphate were similar to those obtained in media with potassium phosphate which indicated that yeast extract in rich media could supply sufficient phosphorous for growth at the 2% sugar level (data not shown).
Overnight cultures in RMG medium were either started from single colonies or from glycerol stocks. Optical densities were measured using a Beckman DU-640 spectrophotometer (Beckman Coulter, Inc., Brea, CA) for inoculation. Growth rates were obtained from the Bioscreen C analyzer purchased from Growth Curves USA (Piscataway, NJ). Procedures for inoculation, growth conditions, measurement, recording of final cell densities and calculations used to correct for non-linear response at high cell densities were previously reported [22 (link)]. In brief, log phase cultures of Z. mobilis 8b (a recombinant xylose-utilizing strain of ZM4) were obtained by inoculating overnight cultures in RMG at 30°C and allowing the cells to grow to an OD600 ~ 1.0. Cells were then spun down at 3840 × g, for 10 min at RT and resuspended in RMG or RMX with inhibitor at the desired concentration such that the starting cell density distributed to Bioscreen C microplates after appropriate dilutions with inhibitors was OD600 = 0.05 (~5 × 106 cells/mL=) in a total volume of 300 μL. Incubations were performed at 30°C and absorbance readings were taken every 10 min. Operation of the Bioscreen C and collection of turbidity measurements (OD420–580) were computer automated with EZ Experiment. Data were collected and exported to Microsoft Excel spreadsheets.
Cultures for mini-fermentation studies at 1X MIC were inoculated with Z. mobilis 8b from seed cultures at an OD600 of 1.0 described above, in 4.5 mL of RM medium containing 5% glucose or 5% xylose and inhibitor compounds at a concentration which would cause 100% inhibition of growth rates (1X MIC) in 6 mL HPLC vials at 30°C, 150 rpm, and were vented with an 18 gauge needle and 0.2 micron syringe filter. Samples (0.5 mL) were removed at 0, 24 and 48 hours post inoculation for OD600 and HPLC analysis.
Cultures for aldehyde conversion studies were inoculated with Z. mobilis 8b at an OD600 of 1.0 in 100 mL of RMG containing 5% glucose in 125 mL unbaffled shake flasks containing aldehyde inhibitors at a concentration that would cause 50% inhibition of growth rates at 30°C, 125 rpm. Samples were removed at 0, 24 and 48 hours for HPLC and growth analysis. Flasks containing inhibitor medium without cells were included to assess abiotic loss due to instability or volatility.
Ammonium salts were prepared by titrating the acids with concentrated ammonium hydroxide, except for the anions: sulfate, chloride, nitrate, acetate, phosphate. Calcium formate was prepared by titrating formic acid with lime. Phosphate stocks were made by preparing 1 M stock solutions of monobasic phosphate and titrating the medium to pH of 5.8 with 1 M dibasic solution.
As a consequence of precipitation of monobasic potassium phosphate with calcium, it was not included in medium containing calcium. In this case, 50 mM MES, pH 5.8, was provided to supply some buffering capacity. Growth rates in RMG or RMX with 50 mM MES, pH 5.8, and no potassium phosphate were similar to those obtained in media with potassium phosphate which indicated that yeast extract in rich media could supply sufficient phosphorous for growth at the 2% sugar level (data not shown).
Overnight cultures in RMG medium were either started from single colonies or from glycerol stocks. Optical densities were measured using a Beckman DU-640 spectrophotometer (Beckman Coulter, Inc., Brea, CA) for inoculation. Growth rates were obtained from the Bioscreen C analyzer purchased from Growth Curves USA (Piscataway, NJ). Procedures for inoculation, growth conditions, measurement, recording of final cell densities and calculations used to correct for non-linear response at high cell densities were previously reported [22 (link)]. In brief, log phase cultures of Z. mobilis 8b (a recombinant xylose-utilizing strain of ZM4) were obtained by inoculating overnight cultures in RMG at 30°C and allowing the cells to grow to an OD600 ~ 1.0. Cells were then spun down at 3840 × g, for 10 min at RT and resuspended in RMG or RMX with inhibitor at the desired concentration such that the starting cell density distributed to Bioscreen C microplates after appropriate dilutions with inhibitors was OD600 = 0.05 (~5 × 106 cells/mL=) in a total volume of 300 μL. Incubations were performed at 30°C and absorbance readings were taken every 10 min. Operation of the Bioscreen C and collection of turbidity measurements (OD420–580) were computer automated with EZ Experiment. Data were collected and exported to Microsoft Excel spreadsheets.
Cultures for mini-fermentation studies at 1X MIC were inoculated with Z. mobilis 8b from seed cultures at an OD600 of 1.0 described above, in 4.5 mL of RM medium containing 5% glucose or 5% xylose and inhibitor compounds at a concentration which would cause 100% inhibition of growth rates (1X MIC) in 6 mL HPLC vials at 30°C, 150 rpm, and were vented with an 18 gauge needle and 0.2 micron syringe filter. Samples (0.5 mL) were removed at 0, 24 and 48 hours post inoculation for OD600 and HPLC analysis.
Cultures for aldehyde conversion studies were inoculated with Z. mobilis 8b at an OD600 of 1.0 in 100 mL of RMG containing 5% glucose in 125 mL unbaffled shake flasks containing aldehyde inhibitors at a concentration that would cause 50% inhibition of growth rates at 30°C, 125 rpm. Samples were removed at 0, 24 and 48 hours for HPLC and growth analysis. Flasks containing inhibitor medium without cells were included to assess abiotic loss due to instability or volatility.
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