deionized water, acid or alkaline solution to form slurry with a
solid: liquid ratio of 1: 5 (in the later study on biomass loading,
greater quantities were used but the solid: liquid ratio was
maintained constant). All the bottles were sealed and crimped
before pretreatment. Acid and alkaline pretreatments were
conducted in an 85 C water bath while thermal pretreatment
(120 C) was conducted in an autoclave. After pretreatment,
the bottle contents were neutralized with a concentrated
H2SO4 or NaOH solution and allowed to cool to ambient temperature
(21 C). The solids content was measured to determine
the loss of the suspended matter during pretreatment.
The COD of the raw biomass, pretreated biomass, and the
liquid fraction of the pretreated feedstock were analyzed. The
best pretreatment conditions were determined based on the
biohydrogen production in ensuing fermentations.