Halophiles are key microorganisms to potentially overcome the salinity-induced limitation in anaerobic bioremediation of organic pollutants and energy recovery when using conventional inoculums. This study aimed to enrich halophiles from hypersaline sediment and optimize their use as alternative inoculum for fermentative hydrogen production. Batch assays of 15 combinations (i.e., pH 6–10, salinity of 10–70 g-NaCl/L, and iron concentration of 0.2–1.0 g-FeSO4/L) were conducted using glucose as a substrate. To potentially boost the efficiency via optimization of operational conditions, response surface methodology (RSM) was employed. The maximum obtained hydrogen yield (HY) of 151 mL/g-COD (1.29 mol-H2/mol-glucose) was achieved at pH 6.0, 40 g-NaCL/L, and 0.6 g-FeSO4/L. The results further emphasize the negative impacts of salinity <25 g-NaCl/L and pH > 8, along with the beneficial role of iron supplementation (0.3–1.0 g-FeSO4/L). By using and validating a multivariate optimization model using desirability function, towards maximum HY, the predicted value was 155 mL/g-COD at pH 6.65, 70 g-NaCl/L, and 0.2 g-FeSO4/L. The microbial community analysis revealed that the enrichment of raw sediment enhanced the biodiversity, and then showed that Halobacteroidaceae bacteria were the key players in producing H2 from glucose. Overall, with respect to the achieved competitive HY (1.29 mol/mol-glucose), the use of halophiles-based inoculum is feasible to overcome the limitations of DF when treating saline wastewater, and future studies/implications with real effluents are worthy.
