For the initial investigations on duckweed pretreatment, fermentations
were carried out in 250 mL glass bottles at 35 C for
5 days. The inoculum for fermentation was collected from a
lab-scale anaerobic digester operated at mesophilic temperature.
Heat shock of fermentation sludge has been reported
effective in preventing hydrogenotrophic methanogenesis
[24,25]. Therefore, the inoculum used in this study was heattreated
at 65 C for 20 min as described by Park et al. [26]
before being mixed with the substrate. The pretreated and
neutralized feedstock (1 g duckweed and pretreatment liquid
volume) was mixed with 2 mL of inoculum, and DI water was
added to the bottle until the total volume of slurry reached
100 mL, leaving a headspace of 150 mL for biogas collection.
The headspace was flushed with nitrogen for 5 min and then
all the bottles were sealed and placed in a 35 C water bath for
fermentation. Throughout the 5 day (static) fermentation, gas
pressure was measured once a day using a digital manometer
fitted with a syringe needle to determine biogas production,
while gas samples (500 mL) were taken for hydrogen analysis.
Subsequent fermentation experiments were conducted
using the best pretreatment method and the conditions in
Table 2. Since temperature and pH were reported to be critical
parameters in the efficiency of biohydrogen production
[26,27], a factorial experiment was used to evaluate their impacts.
Then, a study on biomass loading followed to evaluate
high solids/high salts concentration using the optimum
combination of temperature and initial pH determined previously.
Increasing biomass loading would reduce the size of
the fermenter and ancillary equipment, thus lowering the
capital and operating costs of the process. At higher biomass