Improving biohydrogen production through dark fermentation of steam-heated acid pretreated Alternanthera philoxeroides by mutant Enterobacter aerogenes ZJU1

Alternanthera philoxeroides, a notorious invasive aquatic weed, is a typical lignocellulosic feedstock for fermentative biohydrogen production. To improve the dark fermentation performance, steam-heated acid pretreatment and enzymolysis were employed to release reducing sugars from A. philoxeroides, and Enterobacter aerogenes ZJU1 mutagenized by ⁶⁰Co-γ irradiation was used as the inoculum. Dilute acid accompanied by steam heating significantly disrupted the fiber structures of A. philoxeroides. Scanning electron microscopic images revealed that many pores and fissures were generated in the surface of A. philoxeroides after pretreatment. X-ray diffraction and Fourier transform infrared spectroscopy analyses showed that the pretreatment facilitated the transformation of cellulose I to cellulose II in A. philoxeroides biomass, resulting in the increase of amorphous regions and the decrease of crystallinity. Under the optimum pretreatment condition (1.0 v/v% H₂SO₄, 135 °C for 15 min), the reducing sugar yield reached 0.354 g/g A. philoxeroides, which was further increased to 0.575 g/g A. philoxeroides after enzymolysis. The biohydrogen yield increased by 59.9% from 38.9 mL/g volatile solids (VS) of raw A. philoxeroides to 62.2 mL/gVS of the pretreated one. As compared to the wild strain, E. aerogenes ZJU1 contributed to an increase of 31.8% in the biohydrogen yield from pretreated A. philoxeroides. Further optimization of bacteria suspensions significantly increased the maximum biohydrogen production rate from 1.42 to 4.64 mL/gVS/h, advanced the biohydrogen production peak, and resulted in an increase of 42.8% in biohydrogen yield to 89.8 mL/gVS.