Effect of initial pH,nutrients and temperature on hydrogen production from palm oil mill effluent using thermotolerant consortia and corresponding microbial communities

Thermotolerant consortia were obtained by heat-shock treatment on seed sludge from palm oil mill. Effect of the initial pH (4.5–6.5) on fermentative hydrogen production palm oil mill effluent (POME) showed the optimum pH at 6.0, with the maximum hydrogen production potential of 702.52 mL/L-POME, production rate of 74.54 mL/L/h. Nutrients optimization was investigated by response surface methodology with central composite design (CCD). The optimum nutrients contained 0.25 g urea/L, 0.02 g Na₂HPO₄/L and 0.36 g FeSO₄·7H₂O/L, giving the predicted value of hydrogen production of 1075 mL/L-POME. Validation experiment revealed the actual hydrogen production of 968 mL/L-POME. Studies on the effect of temperature (25–55 °C) revealed that the maximum hydrogen production potential (985.3 mL/L-POME), hydrogen production rate (75.99 mL/L/h) and hydrogen yield (27.09 mL/g COD) were achieved at 55, 45 and 37 °C, respectively. Corresponding microbial community determined by the DGGE profile demonstrated that Clostridium spp. was the dominant species. Clostridium paraputrificum was the only dominant bacterium presented in all temperatures tested, indicating that the strain was thermotolerant.     

Microbial community analysis of thermophilic mixed culture sludge for biohydrogen production from palm oil mill effluent

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.     

Impacts of nano-metal oxides on hydrogen production in anaerobic digestion of palm oil mill effluent – A novel approach

In the present study, hydrogen production from palm oil mill effluent (POME) was investigated with the incorporation of nanoparticles (NPs) comprising of nickel (NiO) and cobalt oxides (CoO). The NPs of NiO and CoO were prepared using hydrothermal method and were further applied to analyse, their effect on hydrogen production. The results demonstrated that, a maxima volumetric hydrogen production rate of 21 ml H₂/L-POME/h with the hydrogen yield of 0.563 L H₂/g-COD_removed was obtained with 1.5 mg/L concentration of NiO NPs. On the other hand, the addition of CoO NPs produced maximum volumetric hydrogen production rate of 18 ml H₂/L-POME/h with a hydrogen yield of 0.487 L H₂/g-COD_removed with 1.0 mg/L of CoO NPs. Results showed that addition of optimal concentration of NiO and CoO NPs to the POME enhances the hydrogen yield by 1.51 and 1.67 fold respectively. Besides, this addition of NiO and CoO enhanced the COD removal efficiency by 15 and 10% respectively as compared to an un-additive NPs POME. The toxicity of NPs was also tested using bacterial viability test, which revealed that application of 3.0 mg/L of NiO and CoO NPs to modified Luria-Bertani (LB) medium had 63% and 83% reduction in bacterial cell growth. The results concluded that supplementation of NiO and CoO NPs under an optimal range to the wastewater can improve the hydrogen productivity.     

Effects of biomass,COD and bicarbonate concentrations on fermentative hydrogen production from POME by granulated sludge in a batch culture

Effects of three selected variables viz. biomass concentration, initial chemical oxygen demand (COD) concentration and initial bicarbonate alkalinity (BA) on biological hydrogen production from palm oil mill effluent (POME) using the granulated sludge in batch culture were investigated. The experimental results were analyzed and modeled using a central composite design (CCD) of response surface methodology (RSM). In order to carry out a comprehensive analysis of the biohydrogen production process, indicative parameters namely hydrogen yield (Y_H), specific hydrogen production rate (SHPR), and COD removal efficiency were studied as the process responses. Maximum hydrogen yield (124.5 mmol H₂/g COD_removed) and specific hydrogen production rate (55.42 mmol H₂/g VSS.d) were achieved at COD_in 3000 and 6500 mg/l, MLVSS 4000 and 2000 mg/l, and initial BA 1100 mg CaCO₃/l, respectively.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l⁻¹ d⁻¹ gave a maximum hydrogen yield of 0.27 l H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ l⁻¹ d⁻¹ (16.9 mmol l⁻¹ h⁻¹). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l⁻¹ d⁻¹) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).     

Pilot-scale of biohythane production from palm oil mill effluent by two-stage thermophilic anaerobic fermentation

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.     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

Application of immobilized upflow anaerobic sludge blanket reactor using Clostridium LS2 for enhanced biohydrogen production and treatment efficiency of palm oil mill effluent

Polyethylene glycol (PEG) gel was used to immobilize hydrogen producing Clostridium LS2 bacteria for hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. The UASB reactor with a PEG-immobilized cell packing ratio of 10% weight to volume ratio (w/v) was optimal for dark hydrogen production. The performance of the UASB reactor fed with palm oil mill effluent (POME) as a carbon source was examined under various hydraulic retention time (HRT) and POME concentration. The best volumetric hydrogen production rate of 365 mL H₂/L/h (or 16.2 mmol/L/h) with a hydrogen yield of 0.38 L H₂/g COD_added was obtained at POME concentration of 30 g COD/L and HRT of 16 h. The average hydrogen content of biogas and COD reduction were 68% and 65%, respectively. The primary soluble metabolites were butyric acid and acetic acid with smaller quantities of other volatile fatty acid and alcohols formed during hydrogen fermentation. More importantly, the feasibility of PEG-immobilized cell UASB reactor for the enhancement of the dark-hydrogen production and treatment of wastewater is demonstrated.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

Ultrasonic pretreatment of palm oil mill effluent: Impact on biohydrogen production,bioelectricity generation,and underlying microbial communities

Substrate bioavailabity is one of the critical factors that determine the relative biohydrogen (bioH₂) yield in fermentative hydrogen production and bioelectricity output in a microbial fuel cell (MFC). In the present undertaking, batch bioH₂ production and MFC-based biolectricity generation from ultrasonically pretreated palm oil mill effluent (POME) were investigated using heat-pretreated anaerobic sludge as seed inoculum. Maximum bioH₂ production (0.7 mmol H₂/g COD) and COD removal (65%) was achieved at pH 7, for POME which was ultrasonically pretreated at a dose of 195 J/mL. Maximum value for bioH₂ productivity and COD removal at this sonication dose was higher by 38% and 20%, respectively, than unsonicated treatments. In batch MFC experiments, the same ultrasound dose led to reduced lag-time in bioelectricity generation with concomitant 25% increase in bioelectricity output (18.3 W/m³) and an increase of COD removal from 30% to 54%, as compared to controls. Quantitative polymerase chain reaction (qPCR) tests on sludge samples from batch bioH₂ production reflected an abundance of gene fragments coding for both clostridial and thermoanaerobacterial [FeFe]-hydrogenase. Fluorescence in situ hybridization (FISH) tests on sludge from MFC experiments showed Clostridium spp. and Thermoanaerobacterium spp. as the dominant microflora. Results suggest the potential of ultrasonicated POME as sustainable feedstock for dark fermentation-based bioH₂ production and MFC-based bioelectricity generation.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Effects of process,operational and environmental variables on biohydrogen production using palm oil mill effluent (POME)

A batch study for biohydrogen production was conducted using raw palm oil mill effluent (POME) and POME sludge as a feed and inoculum respectively. Response Surface Methodology (RSM) was used to design the experiments. Experiments were conducted at different reaction temperatures (30–50 °C), inoculum size to substrate ratios (I:S) and reaction times (8–24 h). An optimum condition of biohydrogen production was achieved with COD removal efficiency of 21.95% with hydrogen yield of 28.47 ml H₂ g^?1 COD removed. The I:S ratio was 40:60, with reaction temperature of 50 °C at 8 h of reaction time. The study showed that a lower substrate concentration (less than 20 g L^?1) for biohydrogen production using pre-settled POME was achievable, with optimum HRT of 8 h under thermophilic condition (50 °C). This study also found that pre-settled POME is feasible to be used as a substrate for biohydrogen production under thermophilic condition.     

Bioaugmentation with Thermoanaerobacterium thermosaccharolyticum W16 to enhance thermophilic hydrogen production using corn stover hydrolysate

In the present work, with corn stover hydrolysate as the substrate, an efficient hydrogen-producing thermophile, Thermoanaerobacterium thermosaccharolyticum W16, was added to three kinds of seed sludge (rotten corn stover (RCS), cow dung compost (CDC), and sludge from anaerobic digestion (SAD)) to investigate the effect of bioaugmentation on thermophilic hydrogen production. Batch test results indicate that the bioaugmentation with a small amount of the strain T. thermosaccharolyticum W16 (5% of total microbes) increased the hydrogen yield to varying degrees (RCS: from 8.78 to 9.90 mmol H₂/g utilized sugar; CDC: from 8.18 to 8.42 mmol H₂/g utilized sugar; SAD: from 8.55 to 9.17 mmol H₂/g utilized sugar). The bioaugmentation process also influenced the soluble metabolites composition towards more acetate and less butyrate production for RCS, and more acetate and less ethanol accumulation for SAD. Microbial community analysis indicates that Thermoanaerobacterium spp. and Clostridium spp. dominated microbial community in all situations and might be mainly responsible for thermophilic hydrogen generation. For RCS and SAD, the bioaugmentation obviously increased the relative abundance of the strain T. thermosaccharolyticum W16 in microbial community, which might be the main reason for the improvement of hydrogen production in these cases.     

Ozonation aided mesophilic biohydrogen production from palm oil mill effluent

Ozone pretreatment of palm oil mill effluent (POME) was employed to improve sustrate biodegradability prior to biological H₂ production. The H₂ production was conducted at varing pHs from 4.0 to 6.0 to examine the impact of pH on the H₂ mesophilic production (37 °C). The optimal pH for H₂ production was 6.0 for both raw and ozonated POME. The POME concentrations were greatly influenced the yields and rates of H₂ production. At the optimal pH, the maximum H₂ production yield of 182 ± 7.2 mL.g⁻¹ COD (7.96 mmoL.g⁻¹ COD) was achieved at the ozonated POME concentration of 30,000 mg COD.L⁻¹. The maximum H₂ production rate (R_max) of 43.1 ± 2.5 mL.h⁻¹ was obtained at the ozonated POME concentration of 25,000 mg COD.L⁻¹. The highest total COD removal was 44% at of 15,000 mg COD.L⁻¹ ozonated POME. Acetic and butyric acids were dominant products during H₂ fermentation and tended to increase with the increased POME concentrations. Ozonation as a pretreatment process showed significant enhancement of the POME biodegradability , and subsequently improved the H₂ production H₂.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Biohydrogen production from palm oil mill effluent (POME) by two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of carbon sources in POME

The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H₂/mol-sugar and 8.54 mmol H₂/L·h respectively, while for the mesophilic stage were 1.19 mol H₂/mol-sugar and 1.47 mmolH₂/L·h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H₂/L·h to 10.34 mmol H₂/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 °C) and mesophilic (37.0 °C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique.     

Thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19: Optimization of media composition

The composition of media for thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19 were optimized in order to maximize the hydrogen production potential (P_s). Results from Plackett-Burman design indicated that FeSO₄, CaCl₂, NaHCO₃, and MgCl₂ had a significantly effect (P ≤ 0.05) on P_s. The optimum media composition obtained from the response surface methodology (RSM) with central composite design (CCD), using the hydrolysate at a total sugar concentration of 8.98 g/L, were (all in mg/L): FeSO₄, 1454.65; MgCl_2, 511.36; CaCl_2, 278.62; and NaHCO_3, 2186.41 in which the P_s of 2397 mL H₂/L were obtained. Verification experiment using the optimum media composition in a continuous stirred tank reactor indicated a highly reproducible result in which the P_s of 2608 mL H₂/L was achieved at a hydraulic retention time of 32 h. The results demonstrated that the media composition obtained from the batch experiment using RSM with CCD can be practically applied to continuously produce hydrogen from the hydrolysate with the least error.     

Co-digestion of oil palm trunk hydrolysate with slaughterhouse wastewater for thermophilic bio-hydrogen production by Thermoanaerobacterium thermosaccharolyticm KKU19

The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H₂SO₄ method using 1.56% (w/v) H₂SO₄ and 7.50 min reaction time at 450 W. The Plackett–Burman method was used to screen the key factors that influenced the hydrogen production potential (P_s). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO₄ concentration influenced the P_s. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum P_s of 2604 ± 86 mL H₂/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO₄ concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The P_s obtained when the hydrolysate was supplemented with SHW (2604mL ± 86 mL H₂/L substrate) was comparable to the P_s obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 ± 87 mL H₂/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source.     

Improvement of biohythane production from Chlorella sp. TISTR 8411 biomass by co-digestion with organic wastes in a two-stage fermentation

The suitability of molasses, Napier grass (Pennisetum purpureum), empty fruit bunches (EFB), palm oil mill effluent (POME), and glycerol waste as a co-substrate with Chlorella sp. TISTR 8411 biomass for biohythane production was investigated. Mono-digestion of Chlorella biomass had hydrogen and methane yield of 23–35 and 164–177 mL gVS⁻¹, respectively. Co-digestion of Chlorella biomass with 2–6% TS of organic wastes was optimized for biohythane production with hydrogen and methane yield of 17–75 and 214–577 mL gVS⁻¹, respectively. The hydrogen and methane yield from co-digestion of Chlorella biomass with molasses, POME, and glycerol waste was increased by 8–100% and 80–264%, respectively. The biohythane production of co-digestion of Chlorella was 6–11 L L-mixed waste⁻¹ with an optimal C/N ratio range of 19–41 and H₂/CH₄ ratio range of 0.06–0.3. Co-digestion of Chlorella biomass was significantly improved biohythane production in term of yield, production rate, and kinetics.     

Thermophilic biohydrogen recovery from palm oil mill effluent

Enhancement of biological H₂ production efficiency with pre-ozonation process of palm oil mill effluent (POME) prior to thermophilic dark fermentation (55 °C) was investigated. H₂ fermentation experiments were conducted using varying concentrations of raw and ozonated POME. Results revealed that H₂ can be produced from both raw and ozonated POME under thermophilic fermentation. Maximum H₂ production yield of 77 mL.g⁻¹COD_removed was obtained from ozonated POME, which was higher than that of 51 mL·g⁻¹ COD_removed obtained from raw POME at the highest concentration of 35,000 mg COD.L⁻¹. Meanwhile, the specific H₂ production rate (R'_max) of 1.9 and 1.5 mL·h⁻¹·g⁻¹ TVS were observed in raw and ozonated POME at the concentration of 25,000 mg COD.L⁻¹, respectively. The main metabolic products during POME fermentation were acetic and butyric acids and trace amount of valeric acid. Propionic acid and ethanol have contributed, which could be reduced H₂ production in all batch experiments for both POME. The highest efficiency of total and soluble COD removal of 24 and 25% was obtained from the raw POME, and those of 19 and 25% was obtained from the ozonated POME. The present study demonstrates that the POME loading was greatly influenced on the H₂ production yields and rates. The comparative results showed that the ozonated POME gave higher H₂ yields than the raw POME. Thus, demonstrating that the ozonation process significantly improved the POME biodegradability, which is able to enhance H₂ production yields. However, the ozone pre-treatment was not improved in the specific H₂ production rates.