Five individual pretreatment methods,including three widely-used protocols (heat,acid and base) and two novel attempts (ultrasonic and ultraviolet),were conducted in batch tests to compare their effects on mixed microflora to enhance hydrogen (H2) production from corn stover hydrolysate.Experimental results indicated that heat and base pretreatments significantly increased H2 yield with the values of 5.03 and 4.45 mmol H2/g sugar utifized,respectively,followed by acid pretreatment of 3.21 mmol H2/g sugar utilized.However,compared with the control (2.70 mmol H2/g sugar utilized),ultrasonic and ultraviolet pretreatments caused indistinctive effects on H2 production with the values of 2.92 and 2.87 mmol H2/g sugar utilized,respectively.The changes of soluble metabolites composition caused by pretreatment were in accordance with H2-producing behavior.Concretely,more acetate accumulation and less ethanol production were found in pretreated processes,meaning that more reduced nicotinamide adenine dinucleotide (NADH) might be saved and flowed into H2-producing pathways.PCR-DGGE analysis indicated that the pretreatment led to the enrichment of some species,which appeared in large amounts and even dominated the microbial community.Most of the dominated species were affiliated to Enterobacter spp.and Escherichia spp.As another efficient H2 producer,Clostridium bifermentan was only found in a large quantity after heat pretreatment.This strain might be mainly responsible for better performance of H2 production in this case.