Analytical methods
Samples from the reactors were immediately filtered on 0.45 µm polyvinylidene fluoride membranes (Millipore, USA) and stored at -20°C until analysis. Volatile fatty acids (VFAs; formate to valerate), lactate, succinate and glucose were analysed using high performance liquid chromatograph (HPLC) method described previously (Rombouts et al., 2019). Ethanol was analysed using a gas chromatography (GC) method described previously (Rombouts et al., 2019). The off-gases were monitored on-line for H2 and CO2 by a connection to a NGA 2000 MLT 1 Multicomponent analyser (Rosemount, Shakopee, Minnesota, USA). Data acquisition (base, H2, CO2) was made using a BBI systems MFCS/win 2.1 (Sartorius, Göttingen, Germany).
Methane was measured manually using GC with a Varian CP 3800 (Varian Medical Systems, Palo Alto, California, USA) equipped with a MolSieve capillary column (1.2 m x 1 mm; 13 x 80/100 mesh, 50 °C) and a thermal conductivity detector (200 °C) with N2 as a carrier gas (2 mL min-1).
Biomass concentration was measured using a standard method which relies on centrifugation of 150 mL to separate the cells from the medium, drying these solids to obtain the total suspended solids (TSS) and burning these solids at 550 °C to determine the amount of volatile suspended solids (VSS) (APHA, 1998). This analysis was coupled to absorbance measurement at 660 nm to establish a correlation. Absorbance values were used to calculate the biomass concentration during the cycle analysis and batch experiments.