Bioreactor enrichment
Both enrichments were performed in 3 L jacketed bioreactors with working
volumes of 2 L. pH was maintained at 5.0 ± 0.1 using NaOH at 4 mol
L-1 and HCl at 1 mol L-1.
Temperature was maintained at 30°C ± 0.1. The cultures were stirred
constantly at 300 rpm. Anaerobic conditions were maintained by sparging
the reactor with a flow of 576 mmol N2
h-1. The off-gas was cooled and dried at 5°C using a
gas condenser. A hydraulic retention time (HRT) and solid retention time
(SRT) of 12 h was maintained by removing 1 L of culture per cycle under
continuous stirring and a cycle time set to 6 h.
The mineral cultivation medium was identical to the one used by Temudo
et al. (2007), while the complex medium was supplemented by 9 B vitamins
and peptides according to Table S1 in the supplementary information. The
carbon source, peptides and B vitamins and the ammonium, phosphate and
trace elements were fed separately from 12.5 concentrated stock
solutions and diluted using N2-sparged demineralized
water. Connected to the base pump was a pump supplying 3% (v:v)
antifoam C (Sigma Aldrich, Germany), which ensured a flow of 3-5 mL
h-1 or 14-17 mL cycle-1. The glucose
solution was sterilised at 110°C for 20 min. For the complex medium, the
peptides were sterilised separately at 110°C for 20 min and the B
vitamins were added by filter sterilisation through 0.45 µm and 0.2 µm
polyvinylidene fluoride filters.
The inoculum for all enrichments consisted of sludge taken from an
anaerobic digester of the wastewater treatment plant (WWTP)
Harnaschpolder, The Netherlands. The pH, temperature, and HRT and SRT of
the digester in the WWTP were 7-7.2, 36-38˚C, and 20 days, respectively.
At the beginning of each experiment, the reactor was seeded with
approximately 10 mL of 200 µm filtered inoculum (0.5% of the total
volume). The reactors were gradually moving from 24-h and 12-h cycles in
3 days to the final desired 6-h cycles to maintain a HRT of 12 h. Steady
state was assumed if during a period of at least 5 days little variation
was detected in the product concentrations.