Strict anaerobic techniques were thoroughly applied in this study. Sterile anoxic bottles were prepared as described in Text S2 (Supplementary Materials) prior to medium dispensing and inoculation. The gas volume/liquid volume ratio was maintained at three for all experiments, regardless of the size of the bottle, unless stated otherwise. The experiments were conducted with four biological replicates in the first stage (gas feeding of the anaerobic sludge) and triplicates in the second stage (enrichment in the mineral medium). A detailed chronology of the culture transfers is provided in Table S4 .
The setup in the first stage was assembled in an anaerobic chamber. Serum bottles of 219.5 mL volume were filled with 50 mL degassed inoculum, sealed with butyl rubber stoppers and crimped with aluminum caps. The gas phase of the serum bottles was replaced by H2 (80%) and CO2 (20%). All bottles receiving H2 and CO2 were operated in fed-batch mode and pressurized daily to ~2.2 bar for approximately five months. Bottles containing the inoculum and a nitrogen atmosphere (not pressurized) were used as controls to account for the residual biogas production. Detailed information about headspace flushing and pressurization is given inText S2 .
In the second stage, medium A was used to enrich a particle-free culture by six subsequent culture transfers in fresh medium bottles by inoculating the content of the preceding culture transfer (10%, v/v). One randomly selected replicate from the first stage served as the inoculum to start the bottles for the second stage. Anoxic medium A (45 mL) was dispensed to sterile, anoxic serum bottles and left overnight in an incubator at 37 °C to reduce any oxygen traces that entered the bottles during medium dispensing. Next, the bottles were inoculated with 5 mL culture from the first stage. Biological controls for determining residual biogas production (containing inoculum but with N2 gas phase), as well as sterile controls (not inoculated, but with either H2/CO2 or N2 gas phase), were also set up. The bottles were fed with a gaseous substrate, as described above, and incubated at 37.4 °C in an orbital shaking incubator (IKA KS 4000 ic control, IKA®-Werke GmbH & Co. KG, Biberach an der Riss, Germany) at 200 rpm.
The setup in the first stage was assembled in an anaerobic chamber. Serum bottles of 219.5 mL volume were filled with 50 mL degassed inoculum, sealed with butyl rubber stoppers and crimped with aluminum caps. The gas phase of the serum bottles was replaced by H2 (80%) and CO2 (20%). All bottles receiving H2 and CO2 were operated in fed-batch mode and pressurized daily to ~2.2 bar for approximately five months. Bottles containing the inoculum and a nitrogen atmosphere (not pressurized) were used as controls to account for the residual biogas production. Detailed information about headspace flushing and pressurization is given in
In the second stage, medium A was used to enrich a particle-free culture by six subsequent culture transfers in fresh medium bottles by inoculating the content of the preceding culture transfer (10%, v/v). One randomly selected replicate from the first stage served as the inoculum to start the bottles for the second stage. Anoxic medium A (45 mL) was dispensed to sterile, anoxic serum bottles and left overnight in an incubator at 37 °C to reduce any oxygen traces that entered the bottles during medium dispensing. Next, the bottles were inoculated with 5 mL culture from the first stage. Biological controls for determining residual biogas production (containing inoculum but with N2 gas phase), as well as sterile controls (not inoculated, but with either H2/CO2 or N2 gas phase), were also set up. The bottles were fed with a gaseous substrate, as described above, and incubated at 37.4 °C in an orbital shaking incubator (IKA KS 4000 ic control, IKA®-Werke GmbH & Co. KG, Biberach an der Riss, Germany) at 200 rpm.
Free full text: Click here
