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%.     

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 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).     

Production of methane from ozonated palm oil mill effluent

Methane (CH₄) production from palm oil mill effluent (POME) pre-treated by ozonation was conducted under mesophilic (37 °C) condition. The results demonstrated that methane can be produced from both non-ozonated and ozonated POME at a concentration range of 3,000 to 15,000 mg COD L⁻¹. Methane yield rised 54% when POME was pre-treated by ozonation at POME concentration of 15,000 mg COD L⁻¹. The methane yield increased the POME concentration was increased. At POME above 15,000 mg COD L⁻¹, the methane yield was dropped dramatically. The methane production rates (R_max) and yields exerted similar trend regarding the POME concentration. Accumulation of volatile fatty acids in the reactor posed the drop of methane production. Ozonation pretreatment process of POME can improve the biodegradability of the complex organic matter in POME and enhanced methane yield and rate at POME concentration range of 3,000–15,000 mg COD L⁻¹.     

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.     

Kinetic analysis of biohydrogen production from anaerobically treated POME in bioreactor under optimized condition

In this study, the biohydrogen production from POME was performed under mesophillic conditions by mixed culture in a 2 L bioreactor using the optimized conditions obtained previously. The effect of controlling pH initially or throughout the fermentation was also examined. The fermentation performance was monitored by comparing P, R_m, λ, and P_s in both systems. In this present study, the reactor system showed higher hydrogen production potential values with the utilization of pH control. Hydrogen production potential was increased two folds when the reactor system was equipped with pH control rather than just fixed the initial pH at 5.8. The biohydrogen production under controlled pH occurred after 7 h fermentation resulting in maximum Ps and Rm of 1.32 L/L POME and 0.144 L/L.h, respectively.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Optimization of conditions for hydrogen production from brewery wastewater by anaerobic sludge using desirability function approach

Brewery wastewater was converted H₂ by anaerobic sludge in batch experiments. A three-factor three-level experimental design of Box-Behnken method was adopted to find the optimum H₂ production conditions. The effects of three major influence factors, temperature, pH and brewery wastewater concentration (BWC), on H₂ yield and H₂ maximum production rate (R_max) were evaluated by applying response surface methodology (RSM) integrating a desirability function approach. Desirable H₂ yield and R_max simultaneously were achieved under temperature 35.9 °C, pH 5.95 and BWC 6.05 g/l by a desirability function approach which produced the maximum overall desirability 0.894. Correspondingly, the H₂ yield and R_max were 149.6 ml H₂/g COD and 53.6 ml/h, respectively. The verification test confirms that the optimum H₂ yield and R_max measured were in good agreement with the predicted values, suggesting that the desirability function approach with RSM was a useful technique to get the maximum H₂ yield and R_max simultaneously.     

Optimization of biohydrogen production from sweet sorghum syrup using statistical methods

This study employed statistically based experimental designs to optimize fermentation conditions for hydrogen production from sweet sorghum syrup by anaerobic mixed cultures. Initial screening of important factors influencing hydrogen production, i.e., total sugar, initial pH, nutrient solution, iron (II) sulphate (FeSO₄), peptone and sodium bicarbonate was conducted by the Plackett–Burman method. Results indicated that only FeSO₄ had statistically significant (P ≤ 0.005) influences on specific hydrogen production (P_s) while total sugar and initial pH had an interdependent effect on P_s. Optimal conditions for the maximal P_s were 25 g/L total sugar, 4.75 initial pH and 1.45 g/L FeSO₄ in which P_s of 6897 mL H₂/L was estimated. Estimated optimum conditions revealed only 0.04% difference from the actual P_s of 6864 mL H₂/L which suggested that the optimal conditions obtained can be practically applied to produce hydrogen from sweet sorghum syrup with the least error.     

Dark fermentative hydrogen production from sucrose and molasses

There are many factors affecting the dark fermentative hydrogen production. The interaction of these factors, that is, their combined effects, should be investigated for better design of the systems with stable and higher hydrogen yields. This study aimed to investigate the combined effects of initial substrate, pH, and biomass (or initial substrate to biomass) values on hydrogen production from sucrose and sugar‐beet molasses. Therefore, optimum initial chemical oxygen demand (COD), pH, and volatile suspended solids (VSS) or initial substrate to biomass (VSS) ratio (S/X_o) values leading to the highest dark fermentative hydrogen production were investigated in batch reactors. An experimental design approach (response surface methodology) was used. Results revealed that when sucrose was the substrate, maximum hydrogen production yield (HY) of 2.3 mol H₂/mol sucrose_added was obtained at initial pH of 7 and COD of 10 g/L. Initial S/X_o values studied (4–20 g COD/g VSS) had no effect on HY, while the initial pH was found as the parameter mostly affecting both HY and hydrogen production rate (HPR). When substrate was molasses, initial COD concentration was the only variable affecting HY and HPR. Maximum of both was achieved at 10 g/L initial COD. Initial VSS values studied (2.5–7.5 g/L) had no effect on HPR and HY. This study also indicated that molasses leads to homoacetogenesis for potentially containing intrinsic microorganism and/or natural constituents; thus, sucrose is more advantageous for hydrogen production via fermentation. Homoacetogenesis should be prevented for effective optimization via response surface methodology, if substrate is a natural carbon source potential to have intrinsic microorganisms. Copyright © 2017 John Wiley & Sons, Ltd.     

Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6

A local bacterial isolate from palm oil mill effluent (POME) sludge, identified as Clostridium butyricum EB6, was used for biohydrogen production. Optimization of biohydrogen production was performed via statistical analysis, namely response surface methodology (RSM), with respect to pH, glucose and iron concentration. The results show that pH, glucose concentration and iron concentration significantly influenced the biohydrogen gas production individually, interactively and quadratically (P < 0.05). The center composite design (CCD) results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO₄ were the optimal conditions for biohydrogen production, yielding 2.2 mol H₂/mol glucose. In confirmation of the experimental model, t-test results showed that curve fitted to the experimental data had a high confidence level, at 95% with t = 2.225. Based on the results of this study, optimization of the culture conditions for C. butyricum EB6 significantly increased the production of biohydrogen.     

Hydrogen production from rice winery wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures

Continuous production of hydrogen from the anaerobic acidogenesis of a high-strength rice winery wastewater by a mixed bacterial flora was demonstrated. The experiment was conducted in a 3.0-l upflow reactor to investigate individual effects of hydraulic retention time (HRT) (2–24 h), chemical oxygen demand (COD) concentration in wastewater (14–36 g COD/l), pH (4.5–6.0) and temperature (20–55°C) on bio-hydrogen production from the wastewater. The biogas produced under all test conditions was composed of mostly hydrogen (53–61%) and carbon dioxide (37–45%), but contained no detectable methane. Specific hydrogen production rate increased with wastewater concentration and temperature, but with a decrease in HRT. An optimum hydrogen production rate of 9.33 lH₂/gVSSd was achieved at an HRT of 2 h, COD of 34 g/l, pH 5.5 and 55°C. The hydrogen yield was in the range of 1.37–2.14 mol/mol-hexose. In addition to acetate, propionate and butyrate, ethanol was also present in the effluent as an aqueous product. The distribution of these compounds in the effluent was more sensitive to wastewater concentration, pH and temperature, but was less sensitive to HRT. This upflow reactor was shown to be a promising biosystem for hydrogen production from high-strength wastewaters by mixed anaerobic cultures.     

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.     

Optimization of biohydrogen production of palm oil mill effluent by ozone pretreatment

The biohydrogen (H₂) production in batch experiments under varying concentrations of raw and ozonated palm oil mill effluent (POME) of 5000–30,000 mg COD.L⁻¹, at initial pH 6, under mesophilic (37 °C), thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. Effects of ozone pretreatment, substrate concentration and fermentation temperature on H₂ production using mesophilic seed sludge was undertaken. The results demonstrated that H₂ can be produced from both raw and ozonated POME, and the amounts of H₂ production were directly increased as the POME concentrations were increased. H₂ was successfully produced under the mesophilic fermentation of ozonated POME, with maximum H₂ yield, and specific H₂ production rate of 182 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 6.2 mL.h⁻¹.g⁻¹ TVS (25,000 mg COD.L⁻¹), respectively. Thus, indicating that the ozone pretreatment could elevate on the biodegradability of major constituents of the POME, which significantly enhanced yields and rates of the H₂ production. H₂ production was not achieved under the thermophilic and extreme-thermophilic fermentation. In both fermentation temperatures with ozonated POME, the maximum H₂ yield was 62 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 63 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹), respectively. The highest efficiency of total and soluble COD removal was obtained at 44 and 37%, respectively following the mesophilic fermentation, of 24 and 25%, respectively under the thermophilic fermentation, of 32 and 20%, respectively under the extreme-thermophilic fermentation. The production of volatile fatty acids increased with an increased fermentation time and temperature in both raw and ozonated POME under all three fermentation temperatures. The accumulation of volatile fatty acids in the reactor content were mostly acetic and butyric acids. H₂ fermentation under the mesophilic condition of 37 °C was the better selection than that of the thermophilic and extreme-thermophilic fermentation.     

Optimizing hydrogen production from organic wastewater treatment in batch reactors through experimental and kinetic analysis

Anaerobic hydrogen production from organic wastewater, an emerging biotechnology to generate clean energy resources from wastewater treatment, is critical for environmental and energy sustainability. In this study, hydrogen production, biomass growth and organic substrate degradation were comprehensively examined at different levels of two critical parameters (chemical oxygen demand (COD) and pH). Hydrogen yields had a reverse correlation with COD concentrations. The highest specific hydrogen yield (SHY) of 2.1 mole H₂/mole glucose was achieved at the lowest COD of 1 g/L and decreased to 0.7 mole H₂/mole glucose at the highest COD of 20 g/L. The pH of 5.5–6.0 was optimal for hydrogen production with the SHY of 1.6 mole H₂/mole glucose, whereas the acidic pH (4.5) and neutral pH (6.0–7.0) lowered the hydrogen yields. Under all operational conditions, acetate and butyrate were the main components in the liquid fermentation products. Additionally, a comprehensive kinetic analysis of biomass growth, substrate degradation and hydrogen production was performed. The maximum rates of microbial growth (μ_m) and substrate utilization (R_su) were 0.03 g biomass/g biomass/day and 0.25 g glucose/g biomass/day, respectively. The optimum pH for the rate of hydrogen production (RH₂$R_{{\text{H}}_2}$) and SHY were 5.89 and 5.74 respectively. Based on the kinetic analysis, the highest RH₂$R_{{\text{H}}_2}$ and SHY for batch-mode anaerobic hydrogen production systems were projected to be 13.7 mL/h and 2.32 mole H₂/mole glucose.     

Behavior of single chambered mediatorless microbial fuel cell (MFC) at acidophilic, neutral and alkaline microenvironments during chemical wastewater treatment

Single chamber mediatorless microbial fuel cell (MFC; non-catalyzed graphite electrodes; open air cathode) behaviour was evaluated under different pH microenvironments [acidophilic (pH 6), neutral (pH 7) and alkaline (pH 8)] during chemical wastewater treatment employing anaerobic mixed consortia as anodic biocatalyst at room temperature (29 ± 2 °C). The performance was found to depend on the feed pH used. Higher current density was observed at acidophilic conditions [pH 6; 186.34 mA/m²; 100 Ω] compared to neutral [pH 7; 146.00 mA/m²; 100 Ω] and alkaline [pH 8; 135.23 mA/m²; 100 Ω]. On the contrary, substrate degradation was found to be effective at neutral pH conditions (ξ_COD – 58.98%; SDR – 0.67 kg COD/m³-day) followed by alkaline (ξ_COD – 55.76%; SDR – 0.62 kg COD/m³-day) and acidophilic (ξ_COD of 47.80%; SDR 0.58 kg COD/m³-day) conditions studied. However, relatively higher specific power yield was observed at acidophilic microenvironment (46 mW/kg COD_R) compared to neutral (35 mW/kg COD_R) and alkaline (34 mW/kg COD_R) conditions. The behaviour of the MFC was also evaluated employing electron discharge, cyclic voltammetry, cell potentials, Coulombic efficiency and sustainable power analysis. Acidophilic operation showed higher Coulombic efficiency and effective electron discharge at relatively higher resistance compared to neutral and alkaline conditions studied.     

Hydrogen production from alcohol wastewater by an anaerobic sequencing batch reactor under thermophilic operation: Nitrogen and phosphorous uptakes and transformation

The objective of this study was to investigate hydrogen production from alcohol wastewater using an anaerobic sequencing batch reactor (ASBR) under thermophilic operation and at a constant pH of 5.5. Under the optimum COD loading rate of 68 kg/m³d, the produced gas contained 43% H₂ without methane and the system provided a hydrogen yield and specific hydrogen production rate of 130 ml H₂/g COD removed and 2100 ml H₂/l d, respectively, which were much higher than those obtained under the mesophilic operation. Under thermophilic operation, both nitrogen and phosphate uptakes were minimal at the optimum COD loading rate for hydrogen production and most nitrogen uptake was derived from organic nitrogen. Under the thermophilic operation for hydrogen production, the nutrient requirement in terms of COD:N:P was found to be 100:6:0.5, which was much higher than that for the methenogenic step for methane production under both thermophilic and mesophilic operations and for the acidogenic step for hydrogen production under mesophilic operation.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.     

Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions

Hydrogen (H₂) production from cheese processing wastewater via dark anaerobic fermentation was conducted using mixed microbial communities under thermophilic conditions. The effects of varying hydraulic retention time (HRT: 1, 2 and 3.5 days) and especially high organic load rates (OLR: 21, 35 and 47 g chemical oxygen demand (COD)/l/day) on biohydrogen production in a continuous stirred tank reactor were investigated. The biogas contained 5–82% (45% on average) hydrogen and the hydrogen production rate ranged from 0.3 to 7.9 l H₂/l/day (2.5 l/l/day on average). H₂ yields of 22, 15 and 5 mmol/g COD (at a constant influent COD of 40 g/l) were achieved at HRT values of 3.5, 2, and 1 days, respectively. On the other hand, H₂ yields were monitored to be 3, 9 and 6 mmol/g COD, for OLR values of 47, 35 and 21 g COD/l/day, when HRT was kept constant at 1 day. The total measurable volatile fatty acid concentration in the effluent (as a function of influent COD) ranged between 118 and 27,012 mg/l, which was mainly composed of acetic acid, iso-butyric acid, butyric acid, propionic acid, formate and lactate. Ethanol and acetone production was also monitored from time to time.To characterize the microbial community in the bioreactor at different HRTs, DNA in mixed liquor samples was extracted immediately for PCR amplification of 16S RNA gene using eubacterial primers corresponding to 8F and 518R. The PCR product was cloned and subjected to DNA sequencing. The sequencing results were analyzed by using MegaBlast available on NCBI website which showed 99% identity to uncultured Thermoanaerobacteriaceae bacterium.     

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.