Experimental design
Communities were cultured for eight weeks in 500 ml bottles (total
volume with headspace: 600 ml, Duran) using Automated Methane Potential
Test System (AMPTS II, Bioprocess Control Sweden AB to measure
CO2-stripped biogas (called biogas in this paper)
production.
Each replicate was started using 300 mL of AD sample. The communities
were put into the fermenters in full carrying capacity. Communities were
acclimatised in the fermenters for a week in order to make sure any
residual biogas production did not obscure the final results. Starting
with the day one of the second week, each community was fed 2g of feed
composed of 3.53% casein, 1.17% peptone, 1.17% albumen, 47.07%
dextrin and 47.07% sucrose (all compounds – Sigma). Feed was suspended
1:10 in sterile water. At the start of the experiment the community was
supplemented with 0.3 mL 1000x Trace Metal Stock (1
gl-1 FeCl2 . 4H2O, 0.5
gl-1 MnCl2 . 4H2O, 0.3
gl-1 CoCl2 . 4H2O, 0.2
gl-1 ZnCl2, 0.1 gl-1NiSO4 . 6H2O, 0.05
gl-1 Na2MoO4 .
4H2O, 0.02 gl-1H3BO3, 0.008 gl-1Na2 WO4 . 2H2O, 0.006
gl-1 Na2SeO3 .
5H2O, 0.002 gl-1 CuCl2. 2H2O).
The experiment (see Fig. 1) was conducted in two stages, with three
treatment regimens (12 replicates each): Stage one, where we cultivated
12 replicates of the community (‘pre-adaptation’) and stage two –
testing the pre-adaptation success – where we cultivated 12 replicates
of the original community and 12 replicates of the original community
supplemented with 1% (w/w) of the pre-adapted community from stage one.
The second stage was started a week before the first was finished to
starve the communities for a week, such that any residual carbon sources
that could be metabolised, were metabolised and did not interfere with
the later gas measurements. After a week of pre-incubation, 1% (w/w) of
the communities from the first stage were transferred to the adaptation
treatments and 1% w/w of stock community was transferred to the control
treatment.