and used as inoculum for dark fermentation
batch tests using serum bottles (see Table 1). The sludge was preheated
at 70 C for 30 min before inoculation to inactivate or kill
methanogens or other H2-consuming microorganisms. Substrateto-
biomass (S/X) ratio was changed from 1 to 8 g COD/g VSS, and
initial pH was adjusted to 5.5 ± 0.2 using 1 N NaOH and HCl before
the experiments. The required volumes of inoculum and sugar beet
juice (substrate) were adjusted to meet the S/X ratios in a total liquid
of 200 mL in serum bottles. The headspace in serum bottles
was flushed with nitrogen gas (99.9%) for 3 min and the bottles
were capped tightly with rubber stoppers. The bottles were then
placed in an incubator (MaxQ 4000, Thermo Scientific, CA) shaken
at 180 rpm at a constant temperature of 37 C. Serum bottle tests
were conducted in triplicate for each condition. Control test (lacking
substrate) was carried out to account for hydrogen gas production
solely from substrate (Lee et al., 2008, 2009a). The volume of
biogas produced from the serum bottles in batch dark fermentation
tests was measured with water displacement method. The
volume of displaced water within the pre-calibrated cylinder represents
the volume of biogas produced.