Nusara SinbuathongBoonya CharnnokBoonsong SillapacharoenkulChularat SakdaronnarongKing Mongkut's University of Technology North BangkokKasetsart UniversityMahidol UniversityPrince of Songkla University2021-02-032021-02-032020-01-01International Journal of Hydrogen Energy. (2020)036031992-s2.0-85098670429https://repository.li.mahidol.ac.th/handle/20.500.14594/60928© 2020 Hydrogen Energy Publications LLC Methods are investigated to prepare active hydrogen (H2)-producing cultures originating from palm oil mill sludge using dark fermentation. The first successful method that produces potent H2-producing cultures and avoids growing H2-consuming methanogens involves heat pretreatment of the sludge at 100 °C for 2 h and then the sludge sample is shocked in an ice bath for 15 min. Subsequently, a glucose solution rich in nutrients (glucose-based substrate) of 14.80 g chemical oxygen demand (COD)/L is fed in to enrich the H2-producing cultures. The H2 production reaches 78.63% on day 31. The second method involves acid pretreatment of sludge with 10 M hydrochloric acid at pH 3 for 48 h. Glucose-based substrate of 25.47 g COD/L is fed into the system. The H2 production is 69.41% on day 27. For both methods, the H2 production is stable after the H2 content reached its maximum. The operation is performed semi-continuously using a hydraulic retention time of 1 day and at 30 °C. The optimum bacterial cells-to-COD level of substrate is approximately 0.60 in the start-up medium. The fermentation medium has an optimum initial pH of 5 and a final pH of 5.2–5.3. These two methods are recommended to produce active H2-producing cultures for plant start-up in bio-H2 production.Mahidol UniversityEnergyPhysics and AstronomyArticleSCOPUS10.1016/j.ijhydene.2020.12.070