Optimization of combined (acid + thermal) pretreatment for enhanced dark fermentative H2 production from Chlorella vulgaris using response surface methodology

This study was performed to optimize the combined (acid + thermal) pretreatment of Chlorella vulgaris for enhanced dark fermentative H2 production. Using response surface methodology, the maximum H2 yield of 47.7 mL H2/g dry cell weight (dcw) was predicted at the optimum conditions of HCl concentration (1.0%), heating temperature (92 °C), and reaction time (47 min). In a confirmation test, the similar result of 48.4 mL H2/g dcw was obtained at the optimum conditions. Meanwhile, only sucrose of 13 g/g dcw was detected from C. vulgaris without pretreatment, while glucose of 21 g/g dcw, fructose of 3 g/g dcw, and maltose of 16 g/g dcw were found from C. vulgaris with combined pretreatment. Fluorescence in situ hybridization (FISH) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses revealed that Clostridium sp. cluster I such as Clostridium butyricum and C. perfringens accounted for 71.7% of total bacteria in a reactor with combined pretreatment.