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Highlights • Combined ultrasound-acid pretreatment improved H 2 yield of grass by 311.7%. • The combined pretreatment improved the substrate utilization efficiency. • The combined pretreatment induced a more efficient fermentation pathway. • More enrichment of Clostridium contributed to the improvement in H 2 production. Abstract In this study, the dilute acid pretreatment combined with ultrasound was applied to improve fermentative hydrogen production from grass. The experimental results indicated that SCOD and soluble carbohydrate contents of grass was improved by 98.6% and 236.9% after the combined treatment, respectively. Surface morphology (SEM and AFM) and crystallinity analysis revealed that the combined pretreatment process could effectively destroyed the biomass structure and increased their surface area. Owing to the increased soluble organics proportion and better enzymatic accessibility of residual solids, the hydrogen yield reached 42.2 mL/g-dry grass after the combined treatment, which was 311.7%, 190.0% and 35.0% higher in comparison with the control, individual ultrasound and acid pretreated groups, respectively. Meanwhile, the combined treatment also increased the substrate utilization efficiency and induced a more efficient fermentation pathway. Bacterial community analysis revealed that more enrichment of Clostridium and less enrichment of Enterococcus contributed to the improved hydrogen production. [ABSTRACT FROM AUTHOR]

Biohydrogen production from sewage sludge through anaerobic fermentation is a cleaner production process. However, the complex sludge structure, especially the outer-layer extracellular polymeric substances, significantly restricts the utilization of available organics in the inner-layer sludge cells for hydrogen production. In view of this, this study firstly adopted a cation-binding agent, sodium citrate (SC), to strip the outer-layer extracellular polymeric substances from the sludge floc, followed by thermal disintegration of microbial cells, which aimed to promote the disintegration performance and biohydrogen productivity. The results showed that the sludge with the combined SC-Thermal pretreatment (0.3 g SC/g-SS + 121 °C for 30 min) achieved the maximum biohydrogen yield of 28.6 mL/g-VS added , which was 346.9%, 73.3% and 148.7% higher that of the control, sole SC pretreated system, and sole thermal pretreated system, respectively. The mechanism study revealed that the combined SC-Thermal pretreatment synergistically disintegrated the sludge floc and promoted the soluble organics content by 107.5 times. Meanwhile, the combined pretreatment also shortened the lag time of hydrogen formation, accelerated the hydrolysis, and improved the organics utilization, thus contributing to the improvement of hydrogen yield. The bacterial community analysis demonstrated that the combined pretreatment promoted the abundances of hydrolytic genera (Bacteroides , Proteocatella and Proteiniclasticum) and hydrogen-producing genera (Clostridium sensu stricto , Citrobacter , Paraclostridium and Escherichia-Shigella), which might be fundamentally responsible for the improvement of biohydrogen productivity. [Display omitted] • Combined sodium citrate-thermal pretreatment evidently disrupted the sludge flocs • Pretreatment promoted the SCOD content by 107.5 times and accelerated the hydrolysis • Pretreatment improved the bioH 2 yield by 346.9% and shortened the lag phase • Pretreated sludge exhibited the mixed acid type fermentation • Pretreatment improved the abundance of hydrogen-producing bacteria in the reactor [ABSTRACT FROM AUTHOR]