C. hydrogenoformans (DSM 6008) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The strain was cultivated in shake-culture at 100 rpm under strictly anaerobic conditions at 70°C in a basal mineral medium buffered with a bicarbonate-phosphate buffer. The medium as formulated by Zhao et al. (2011) contained (in g · L–1 of demineralized water): KCl 0.33, MgCl2 · 6H2O 0.102, CaCl2 · 2H2O 0.015, NH4Cl 0.33, KH2PO4 0.136, NaHCO3 0.42, yeast extract 0.05, Na2S · 9H2O 0.7. The medium was supplemented with 10 mL · L–1 trace metals solution and 10 ml · L–1 of vitamins solution prepared as described previously (Stams et al. 1993). All stock solutions were autoclaved, except for the vitamin solution, which was sterilized by filtration through 0.22 μm filter membranes. The initial pH was adjusted between 6.8 and 7.0.
The pyruvate activity tests were conducted in 120 mL serum bottles in quintuplicate. Bottles were filled with 30 mL of the growth medium, inoculated with 2 mL of C. hydrogenoformans at an initial concentration of 6.65 mg volatile suspended solid (VSS) · L–1, capped, and flushed with a gas mixture of N2/CO2 to establish anaerobic conditions. Starter cultures were active C. hydrogenoformans cultures that were cultivated on CO as sole source of carbon and energy. Headspace monitoring of these cultures was done prior to inoculation to ensure that they were not in latency phase. Bottles were then flushed with a high purity CO gas (100%) and set to atmospheric pressure or spiked with a pyruvate solution to an initial concentration of 3.0 ± 0.1 g · L–1, and incubated in the absence of light at 70°C and 100 rpm in a rotary shaker (New Brunswick, Edison, NJ).
Microbial quantification in the bottles was performed immediately after substrate feeding (time 0), then intermittently until the end of experiment. Biomass quantification was achieved using chemical oxygen demand (COD) measurements which were then converted to VSS using a factor of 1.37 g COD · g–1 VSS based on the elemental formula of microbial biomass as CH1.79O0.5N0.2S0.005 (Roels 1983).
Both substrate (CO, pyruvate) depletion and catabolite (H2, CO2, volatile fatty acids (VFA) and alcohols) production were monitored. The specific substrate uptake or product formation rates, expressed as mol · g–1 VSS · d–1, were obtained by the rate of depletion or accumulation (mol · d–1) per bottle divided by the number of grams of biomass-VSS as estimated in the bottle. The hydrogen yield (YH2) was expressed as a percentage of the H2 gas produced per CO consumed (mol/mol).
The COD was determined according to Standard Methods (Eaton et al. 2005), using a spectrophotometer DRB 200 (Hach Company, Loveland, CO).
Gas samples were obtained by withdrawing 300 μL of gas from the bottle headspace using a gas-tight syringe (model 1750 Hamilton, Reno, NV). Gas composition (H2, CO, CO2) was measured by injecting this gas into a HP 6890 gas chromatograph (Hewlett Packard, Palo Alto, CA) equipped with a thermal conductivity detector (TCD) and a 11 m × 3.2 mm 60/80 mesh Chromosorb 102 packed column (Supelco, Bellafonte, PA). The column temperature was held at 60°C for 7 min and increased to 225°C at a rate of 60°C per min. Argon was used as the carrier gas. The injector and detector were maintained at 125°C and 150°C respectively.
VFAs (i.e. acetic, propionic and butyric acids) were measured on an Agilent 6890 (Wilmington, DE) gas chromatograph (GC) equipped with a flame ionization detector (FID). 0.2 μL samples were diluted 1:1 (vol./vol.) with an internal standard of iso-butyric acid in 6% formic acid, directly injected on a glass column of 1 m × 2 mm Carbopack C (60–80 mesh) coated with 0.3% Carbowax 20M and 0.1% H3PO4. The column was held at 130°C for 4 min. Helium was the carrier gas, fed at a rate of 20 mL · min–1. Both injector and detector were maintained at 200°C.
For measurement of solvents (methanol, ethanol, acetone, 2-propanol, tert-butanol, n-propanol, sec-butanol, n-butanol) 100 μL of liquid was transferred into a vial that had 20 mL of headspace and was crimp sealed with a Teflon-coated septum. The vial was heated at 80°C for 2 min, then 1000 μL of headspace gas was injected onto a DB-ACL2 capillary column of 30 m × 530 mm × 2 μm using a Combipal autosampler (CTC Analytics AG, Zwingen, Switzerland). The column was held at 40°C for 10 min. Helium was the carrier gas at a head pressure of 5 psi. The injector and the detector were maintained at 200°C and 250°C, respectively.
Pyruvate was monitored using a high performance liquid chromatograph (HPLC) (Waters, Milford, MA) equipped with a model 600 pump, a model 717 Plus autosampler and a refractive index detector (model 2414) linked to a photodiode array (PDA) detector (model 2996). The separation was made on a 300 mm × 7.8 mm ICSep IC ION-300 column (Transgenomic, Omaha, NE). The mobile phase was 0.01 N H2SO4 at 0.4 mL · min–1. Analyses were carried out at 35°C.