2.3. Optimization of the key parameters affecting methane
production in the batch experiment
RSM with CCD was used to optimize the key factors affecting
simultaneous methane production and biodegradability. The
investigated parameters included the ratio of substrate to biomass
(S/X ratio) (X1) (g-VSsub/g-VSinoculum), Ni concentration (mg/L) (X2),
and Co concentration (mg/L) (X3). The response variables were MY
and biodegradability (Table 2). The MY was calculated by dividing
the methane production (mL-CH4/L) by the substrate concentration
(g-VSsub/L). Biodegradability was calculated as follows:
Biodegradability (%) ¼ [Methane yield at STP (mL-CH4/g-COD)/
Theoretical methane yield (350 mL-CH4/g-COD)] 100 (1)
Design-Expert (Demo version 7.0, Stat-Ease, Inc., Minneapolis,
MN, USA) was employed for experimental design, modeling, and
graphical display of experimental results.
Production of methane was conducted in 120-mL serum bottles
each having 70-mLof working volume. The medium used to produce
methane contained acidic effluent, and Ni and Co, as well as
the inoculum. Table 2 shows the S/X ratio and concentration of Ni
and Co used in the fermentation. The initial pH of the medium was
adjusted to 7.0 using 2 mol/L of HCl or 2 mol/L of NaOH. The serum
bottles were capped with rubber stoppers and aluminum caps.
They were subsequently flushed with nitrogen gas to ensure
anaerobic conditions. All bottles were incubated at room temperature
(30 ± 2 C) on a shaker operating at 150 rpm. All experiments
were done in triplicates