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Improvement of biohythane production from oil palm industry solid waste residues by co-digestion with palm oil mill effluent (POME) in two-stage thermophilic fermentation was investigated. A two-stage co-digestion of solid waste with POME has biohythane production of 26.5–34 m3/ton waste. The co-digestion of solid waste with POME increased biohythane production of 67–114% compared to digestion POME alone. Co-digestion of solid waste with POME enhanced hydrolysis constant (kh) from 0.07 to 0.113 to 0.120–0.223 d−1. The hydrolysis constant (kh) of co-digestion was 10 times higher than the single digestion of solid waste. Clostridium sp. was predominated in the hydrogen stage, while Methanosphaera sp. was predominant in methane stage. The co-digestion of solid waste with readily biodegradable organic matter (POME) could significantly increase biohythane production with achieving the significant cost reduction for pretreatment of solid wastes.

The pilot-scale of two-stage thermophilic (55 °C) for biohythane production from palm oil mill effluent (POME) was operated at hydraulic retention time (HRT) of 2 days and organic loading rate (OLR) of 27.5 gCOD/L⋅d) for first stage and HRT of 10 days and OLR of 5.5 gCOD/L⋅d for second stage. Biohythane production rate was 1.93 L-gas/L⋅d with biogas containing 11% H2, 37% CO2, and 52% CH4. Recirculation of methane effluent mixed with POME at a ratio of 1:1 can control pH in the first stage at an optimal range of 5.0–6.5. Microbial community in hydrogen stage dominated by Thermoanaerobacterium sp., while methane stage dominated by Methanosarcina sp. The H2/CH4 ratio of biohythane was 0.13–0.18 which suitable for vehicle fuel. Biohythane production from POME could be promising cleaner biofuel with flexible and controllable H2/CH4 ratio.

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L−1, respectively) with maximum H2 and CH4 yields of 139 mL H2 gVS−1 and 454 mL CH4 gVS−1, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H2 stage and enhancing number methanogens in the CH4 stage.

Two-stage thermophilic fermentation and mesophilic methanogenic process with methanogenic effluent recirculation to hydrogen reactor for biohythane production from palm oil mill effluent (POME) was investigated. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 L CH4/L-POME) from POME in batch tests were achieved at 30% recirculation rate of methanogenic effluent. Continuous hydrogen production of without and with recirculation phase was 2.2 and 3.8 L H2/L-POME, respectively. Continuous methane production of without and with recirculation phase was 12.2 and 14 L CH4/L-POME, respectively. Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. Two-stage process with methanogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor and efficiently for energy recovery from POME.

Co-fermentation of palm oil mill effluent (POME) for hydrogen production by Thermoanaerobacterium thermosaccharolyticum PSU-2 under thermophilic condition was investigated. Hydrogen production from co-fermentation POME with 5 g/L decanter cake and without was 2.6 and 1.7 L-H 2 /L-POME. Co-fermentation of POME with decanter cake enhanced hydrogen production. Tbm. thermosaccharolyticum PSU-2 augmentation at 20% v/v was found to be an efficient start-up strategy in continuous hydrogen production from co-fermentation of POME with decanter cake. Tbm. thermosaccharolyticum PSU-2 augmentation could suppress normal flora bacteria in POME and decanter cake. Hydrogen production of 7.2 L-H 2 /L-POME and hydrogen production rate of 18.1 mmol-H 2 /L/h was achieved from continuous hydrogen in long term operation. The soluble metabolites were dominated by acetic acid and butyric acid. Tbm. thermosaccharolyticum PSU-2 augmentation could use for control undesired bacteria in the hydrogen production system with flavored Thermoanaerobacterium sp. as dominant species.

A two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent (POME) was investigated. The hydrogen and methane potential from POME were 170–200 L H 2 kgCOD −1 and 210–292 L CH 4 kgCOD −1 , respectively. A continuous two-stage process with hydraulic retention time (HRT) of 2 d for hydrogen reactor and 15 d for methane reactor, obtained 34% higher energy yield than single stage methane production. The hydrogen and methane yields from two-stage were 210 L H 2 kgCOD −1 and 315 L CH 4 kgCOD −1 , respectively with total energy yield of 15.34 MJ kgCOD −1 . Mixed hydrogen and methane (biohythane) production rate was 4.4 L biogas L −1 d −1 with containing of 51% CH 4 , 14% H 2 and 35% CO 2 . Hydrogen reactor was dominated with hydrogen producing bacteria of Thermoanaerobacterium thermosaccharolyticum , while acetoclastic Methanoculleus sp. was the dominant methanogen in methane reactor. Two-stage process for biohythane production could efficiently for energy recovery from POME.

The microbial community structure of thermophilic mixed culture sludge used for biohydrogen production from palm oil mill effluent was analyzed by fluorescence in situ hybridization (FISH) and 16S rRNA gene clone library techniques. The hydrogen-producing bacteria were isolated and their ability to produce hydrogen was confirmed. The microbial community was dominated by Thermoanaerobacterium species (∼66%). The remaining microorganisms belonged to Clostridium and Desulfotomaculum spp. (∼28% and ∼6%, respectively). Three hydrogen-producing strains, namely HPB-1, HPB-2, and HPB-3, were isolated. 16S rRNA gene sequence analysis of HPB-1 and HPB-2 revealed a high similarity to Thermoanaerobacterium thermosaccharolyticum (98.6% and 99.0%, respectively). The Thermoanaerobacterium HPB-2 strain was a promising candidate for thermophilic fermentative hydrogen production with a hydrogen yield of 2.53 mol H2 mol−1hexose from organic waste and wastewater containing a mixture of hexose and pentose sugars. Thermoanaerobacterium species play a major role in thermophilic hydrogen production as confirmed both by molecular and cultivation-based analyses.

Thermoanaerobacterium-rich sludge was used for hydrogen production and phenol removal from palm oil mill effluent (POME) in the presence of phenol concentration of 100–1000 mg/L. Thermoanaerobacterium-rich sludge yielded the most hydrogen of 4.2 L H2/L-POME with 65% phenol removal efficiency at 400 mg/L phenol. Butyric acid and acetic acid were the main metabolites. The effects of oil palm ash, NH4NO3 and iron concentration (Fe2+) on hydrogen production and phenol removal efficiency from POME by Thermoanaerobacterium-rich sludge was investigated using response surface methodology (RSM). The RSM results indicated that the presence of 0.2 g Fe2+/L, 0.3 g/L NH4NO3 and 20 g/L oil palm ash in POME could improved phenol removal efficiency, with predicted hydrogen production and phenol removal efficiency of 3.45 L H2/L-POME and 93%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with hydrogen production and phenol removal efficiency of 3.43 ± 0.12 L H2/L-POME and 92 ± 1.5%, respectively. Simultaneous hydrogen production and phenol removal efficiency in continuous stirred tank reactor at hydraulic retention time (HRT) of 1 and 2 days were 4.0 L H2/L-POME with 85% and 4.2 L H2/L-POME with 92%, respectively. Phenol degrading Thermoanaerobacterium-rich sludge comprised of Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium aciditolerans, Desulfotomaculum sp., Bacillus coagulans and Clostridium uzonii. Phenol degrading Thermoanaerobacterium-rich sludge has great potential to harvest hydrogen from phenol-containing wastewater.