Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Enhanced hydrogen production from sewage sludge by cofermentation with wine vinasse

The cofermentation of sewage sludge and wine vinasse at different mixing ratios to enhance hydrogen production was investigated. Batch experiments were carried out under thermophilic conditions with thermophilic sludge inoculum obtained from an acidogenic anaerobic reactor. The results showed that the addition of wine vinasse enhances the hydrogen production of sewage sludge fermentation. The highest hydrogen yields, 41.16 ± 3.57 and 43.25 ± 1.52 mL H₂/g VS_added, were obtained at sludge:vinasse ratios of 50:50 and 25:75, respectively. These yields were 13 and 14 times higher than that obtained in the monofermentation of sludge (3.17 ± 1.28 mL H₂/g VS_added). The highest VS removal (37%) was obtained at a mixing ratio of 25:75. Cofermentation had a synergistic effect the hydrogen yield obtained at a sludge:vinasse ratio of 50:50 was 40% higher, comparing to the sum of each waste. Furthermore, kinetic analysis showed that Cone and first-order kinetic models fitted hydrogen production better than the modified Gompertz model.     

Effect of food to microorganisms (F/M) ratio on biohythane production via single-stage dark fermentation

Hythane is a mixture of hydrogen and methane gases which are generally produced in separate ways. This work studied mesophilic biohythane gas (H₂+CH₄+CO₂) production in a bioreactor via single-stage dark fermentation. The fermentation was conducted in batch mode using mixed anaerobic microflora and food waste and condensed molasses fermentation soluble to elucidate the effects of food to microorganisms (F/M) ratio (ranging from 0.2 to 38.2) on gas production, metabolite variation, kinetics and biohythane-composition indicator performances. The experimental results indicate that the F/M ratio and fermentation time affect biohythane production efficiency with values of peak maximum hydrogen production rate 9.60 L/L-d, maximum methane production rate 0.72 L/L-d, and hydrogen yield (HY) of 6.17 mol H₂/kg COD_added. Depending on the F/M ratios, the H₂, CH₄ and CO₂ biogas components were 10–60%, 5–20% and 35–70%, respectively. Prospects for the further real application for single-stage biohythane fermentation based on the experimental data are proposed. This work characterizes an important reactor operation factor F/M ratio for innovative single-stage dark fermentation.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis

A two-stage dark-fermentation and electrohydrogenesis process was used to convert the recalcitrant lignocellulosic materials into hydrogen gas at high yields and rates. Fermentation using Clostridium thermocellum produced 1.67 mol H₂/mol-glucose at a rate of 0.25 L H₂/L-d with a corn stover lignocellulose feed, and 1.64 mol H₂/mol-glucose and 1.65 L H₂/L-d with a cellobiose feed. The lignocelluose and cellobiose fermentation effluent consisted primarily of: acetic, lactic, succinic, and formic acids and ethanol. An additional 800 ± 290 mL H₂/g-COD was produced from a synthetic effluent with a wastewater inoculum (fermentation effluent inoculum; FEI) by electrohydrogensis using microbial electrolysis cells (MECs). Hydrogen yields were increased to 980 ± 110 mL H₂/g-COD with the synthetic effluent by combining in the inoculum samples from multiple microbial fuel cells (MFCs) each pre-acclimated to a single substrate (single substrate inocula; SSI). Hydrogen yields and production rates with SSI and the actual fermentation effluents were 980 ± 110 mL/g-COD and 1.11 ± 0.13 L/L-d (synthetic); 900 ± 140 mL/g-COD and 0.96 ± 0.16 L/L-d (cellobiose); and 750 ± 180 mL/g-COD and 1.00 ± 0.19 L/L-d (lignocellulose). A maximum hydrogen production rate of 1.11 ± 0.13 L H₂/L reactor/d was produced with synthetic effluent. Energy efficiencies based on electricity needed for the MEC using SSI were 270 ± 20% for the synthetic effluent, 230 ± 50% for lignocellulose effluent and 220 ± 30% for the cellobiose effluent. COD removals were ∼90% for the synthetic effluents, and 70–85% based on VFA removal (65% COD removal) with the cellobiose and lignocellulose effluent. The overall hydrogen yield was 9.95 mol-H₂/mol-glucose for the cellobiose. These results show that pre-acclimation of MFCs to single substrates improves performance with a complex mixture of substrates, and that high hydrogen yields and gas production rates can be achieved using a two-stage fermentation and MEC process.     

Fermentative hydrogen production from wastewaters: A review and prognosis

Biohydrogen is a promising candidate which can replace a part of our fossil fuels need in day-to-day life due its perceived environmental benefits and availability through dark fermentation of organic substrates. Moreover, advances in biohydrogen production technologies based on organic wastewater conversion could solve the issues related to food security, climate change, energy security and clean development in the future. An evaluation of studies reported on biohydrogen production from different wastewaters will be of immense importance in economizing production technologies. Here we have reviewed biohydrogen production yields and rates from different wastewaters using sludges and microbial consortiums and evaluated the feasibility of biohydrogen production from unexplored wastewaters and development of integrated bioenergy process. Biohydrogen production has been observed in the range of substrate concentration 0.25–160 g COD/L, pH 4–8, temperature 23–60 °C, HRT 0.5–72 h with various types of reactor configuration. The most efficient hydrogen production has been obtained at an organic loading rate (OLR) 320 g COD/L/d, substrate concentration 40 g COD/L, HRT 3 h, pH 5.5–6.0, temperature 35 °C in a continuously-stirred tank reactor system using mixed cultures and fed with condensed molasses fermentation soluble wastewater. The net energy efficiency analysis showed vinasse wastewater has the highest positive net energy gain followed by glycerin wastewater and domestic sewage as 140.39, 68.65, 51.84 kJ/g COD feedstock with the hydrogen yield (HY) of 10 mmol/g COD respectively.     

Discontinuous biomass recycling as a successful strategy to enhance continuous hydrogen production at high organic loading rates

A novel strategy to discontinuously increase the biomass concentration in a continuous stirred-tank reactor was evaluated to enhance the performance of dark fermentation. Different concentrations of biomass were evaluated at organic loading rates (OLR) ranging from 90 to 160 g lactose/L-d with a hydraulic retention time (HRT) of 6 h. The study revealed that the discontinuous increase of biomass enhanced the hydrogen (H₂) production rates and carboxylic acids concentrations by 19–25% and 8–23%, respectively. In particular, a maximum H₂ production rate of 30.8 L H₂/L-d with carboxylic acids concentration of 20 g/L was reached at an OLR of 138 g lactose/L-d with a biomass concentration of 15 g volatile suspended solids/L. The analysis of microbial communities showed the co-dominance of Clostridium and lactic acid bacteria. Overall, the discontinuous increase of biomass was an effective strategy to improve the performance of suspended-biomass reactors operated at high OLR and low HRT.     

Sequential hydrogen and methane production using the residual biocatalyst of biodiesel synthesis as raw material

Residual Fermented Solid (RFS) is the used biocatalyst obtained after enzymatic biodiesel production carried out applying the fermented solid (FS) with lipase activity. Approximately 350 g of RFS are generated for each liter of biodiesel produced from palm residues fermented solid. In this study, this residue was used for the first time as a raw material for biological hydrogen production through dark fermentation and sequential application of the hydrogen production liquid waste (HPLW) for methane obtainment via anaerobic digestion. The RFS was composed mostly of oils and fats (60% wt.%), and carbohydrates, such as mannose, glucose, and xylose. Hydrogen yield reached 239 ± 44 mL H₂/L after 24 h of fermentation using 31 g_RFS/L at the beginning of the process. Additionally, 204 ± 13 mL CH₄/g COD were produced through the anaerobic digestion of HPLW, which represented 61% of efficiency.     

Hydrogen and methane production via a two-stage processes (H2-SBR + CH4-UASB) using tequila vinasses

The feasibility of producing hydrogen and methane via a two-stage fermentation of tequila vinasses was evaluated in sequencing batch (SBR) and up-flow anaerobic sludge blanket (UASB) reactors. Different vinasses concentrations ranging from 500 mg COD/L to 16 g COD/L were studied in SBR by using thermally pre-treated anaerobic sludge as inoculum for hydrogen production. Peak volumetric hydrogen production rate and specific hydrogen production were attained as 57.4 ± 4.0 mL H₂/L-h and 918 ± 63 mL H₂/gVSS-d, at the substrate concentration of 16 g COD/L and 6 h of hydraulic retention time (HRT). Increasing substrate concentration has no effect on the specific hydrogen production rate. The fermentation effluent was used for methane production in an UASB reactor. The higher methane composition in the biogas was achieved as 68% at an influent concentration of 1636 mg COD/L. Peak methane volumetric, specific production rates and yield were attained as 11.7 ± 0.7 mL CH₄/L-h, 7.2 ± 0.4 mL CH₄/g COD-h and 257.9 ± 13.8 mL CH₄/g COD at 24 h-HRT and a substrate concentration of 1636 mg COD/L. An overall organic matter removal (SBR + UASB) in this two-stage process of 73–75% was achieved.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Evaluation of process performance on biohydrogen production in continuous fixed bed reactor (C-FBR) using acid algae hydrolysate (AAH) as feedstock

In this study, a novel inoculation method to mitigate the inhibition of 5-hydroxymethylfurfural (5-HMF) is proposed. Acid algae hydrolysate containing 1.5 g 5-HMF/L and 15 g hexose/L hexose was fed to a continuous fixed bed reactor (C-FBR) partially packed with hybrid-immobilized beads. The inoculation method enabled a high rate of H₂ production, due to the reduction of 5-HMF inhibition and enhanced biofilm formation. Maximum hydrogen production was achieved at a hydraulic retention time of 6 h with a hydrogen production rate (HPR) of 20.0 ± 3.3 L H₂/L-d and a hydrogen yield (HY) of 2.3 ± 0.4 mol H₂/mol hexose _added. Butyrate and acetate were the major soluble metabolic products released during fermentation. Quantitative real-time polymerase chain reaction analysis revealed that Clostridium butyricum comprised 94.3% of the total bacteria, which was attributed to the high rate of biohydrogen production.     

Effect of system optimizing conditions on biohydrogen production from herbal wastewater by slaughterhouse sludge

The study evaluates the biohydrogen production from herbal wastewater as the substrate by the enriched mixed slaughterhouse sludge as the seed source. In the following experiments, batch-fermentations are carried out with the optimum substrate concentrations, fermentation pH and fermentation temperature to observe the effects of H₂ production, hydrogen yield and other fermentation end products at different conditions. The hydrogen production is increased as substrate concentration increased up to 8 g COD/L WW, but drastically decreased at 10 g COD/L WW. When the pH of fermentation is controlled to 6.5, a maximum amount of hydrogen yield could be obtained. The hydrogen production is maximum at 50 °C (930 ± 30 mL/L WW) compared to 30 °C (436 ± 16 mL/L WW). Acid-forming pathway with butyric acid as a major metabolite dominated the metabolic flow during the hydrogen production. The experimental results indicated that effective hydrogen production from the herbal wastewater could be obtained by thermophilic acidogenesis at proper operational conditions.     

A new side-looking at the dark fermentation of sugarcane vinasse: Improving the carboxylates production in mesophilic EGSB by selection of the hydraulic retention time and substrate concentration

In recent years, a lot of scientific effort has been put into reusing the energy potential of sugarcane vinasse by dark fermentation. However, the findings so far indicate that new pathways need to be followed. In this context, this study assessed the effect of hydraulic retention time (HRT, from 24 to 1 h) on vinasse fermentation (10, 20, and 30 g COD L^?1) in three mesophilic expanded granular sludge bed reactors (EGSB). The carbohydrate conversion remained above 60% at all organic loading rates applied. The maximum hydrogen production rate (8.77 L day^?1 L^?1) was obtained for 720 kg COD m^?3 day^?1 and associated to the lactate-acetate pathway. The highest productivities of propionic, acetic, and butyric acids were 3.11, 1.68, and 2.45 g L^?1 h^?1, respectively, at a HRT of 1 h. At this HRT, the degrees of acidification remained between 54% and 76% in all EGSB reactors. This research provides insights for carboxylate production from sugarcane vinasse and suggests applying the EGSB setup in the acidogenic stage of two-stage processes.     

Microbial ecology of a lactate-driven dark fermentation process producing hydrogen under carbohydrate-limiting conditions

Despite the high prevalence of lactic acid bacteria in dark fermentation (DF) processes, their ecological role is not yet completely elucidated, preventing their systematic use as “helpers” for hydrogen production. The aim of this study was to investigate the microbial community structure of a lactate-driven DF process that successfully produced hydrogen under carbohydrate-limiting conditions using tequila vinasse as a substrate. Microbial responses to stepwise decreases in hydraulic retention time (HRT) from 24 to 4 h were assessed by using Illumina MiSeq sequencing. HRTs above 12 h and below 6 h led to a lower hydrogen production rate (HPR; 0.2–3.3 L/L-d) and process stability (HPR variations within 25–65%), which were associated with the presence of Acetobacter lovaniensis, Clostridium luticellari, Blautia coccoides, and the high abundance of propionate and lactate. Interestingly, transient conditions from unsteady-to-steady state occurred at an HRT of 12 h, where species richness and evenness decreased remarkably. Accordingly, HRTs between 12 and 6 h resulted in higher HPRs of up to 11.7 ± 0.7 L/L-d with HPR variations of less than 10%, which closely matched with the dominance of Clostridium sp., and butyrate and acetate as the main aqueous products. Overall, the results indicate that the successfulness of exploiting the ‘unwanted’ LAB proliferation through lactate-driven DF processes requires the enrichment of lactate-consuming and hydrogen-producing bacteria, which entails the selection of proper biocatalysts and operating conditions/strategies such as the operation of DF reactors under carbohydrate-limiting conditions and low HRTs.     

Anaerobic hydrogen and methane production from low-strength beverage wastewater

In this study, the low-strength effluent from an equalization tank of the wastewater treatment plant in a beverage factory was used for the production of hydrogen and methane. The COD concentration of this low-strength wastewater was 2.9 ± 2.0 g COD/L. In a two-phase anaerobic fermentation system, the hydrogen-producing bioreactor was operated at HRT 8 h, while the methane-producing reactor operated at HRT 24 h. The maximum MPR, methane yield (MY), methane content and COD removal were 72 ± 31 mL/L-d, 58 ± 12 mL/g COD, 92 ± 2% and 78%, respectively. Energy efficiency in this study was calculated as follows, the maximum heating value was 2.2 × 10⁸ kcal/y. The annual carbon-emission reduction was 8.8 × 10⁴ kg CO₂/y, 5.2 × 10⁴ kg CO₂/y, 7.2 × 10⁴ kg CO₂/y when energy-equivalent coal, natural gas or fuel was used, respectively.     

Nickel powder blended activated carbon cathodes for hydrogen production in microbial electrolysis cells

Although pure Ni catalysts can achieve a hydrogen production rate similar to Pt in microbial electrolysis cells (MECs), a reduction in the amount of Ni used is needed to reduce the cost. In this study, nickel powder (pNi) was blended with activated carbon (AC) to reduce the mass of Ni used, while improving catalytic activity for the hydrogen evolution reaction (HER) by increasing the active surface area. Ni powder blended AC cathodes (AC-pNi) were fabricated at different nickel powder loadings (4.8, 19, 46 mg/cm² with AC and 77 mg/cm² without AC as control). AC-pNi4.8 (Ni loading: 4.8 mg/cm²) produced higher hydrogen production rates (0.38 ± 0.04 L-H₂/L-d) than pNi77 (0.28 ± 0.02 L-H₂/L-d) with a 16 times less Ni loading. Cathodic hydrogen recovery of using the AC-pNi4.8 (98 ± 5%) was also higher than pNi77 (82 ± 4%), indicating catalytic activities were improved by AC blending. Nickel dissolution into the catholyte after completion of each cycle was negligible for AC-pNi4.8 (<0.2 mg/L), while Ni dissolution was detected for pNi77 (5–10 mg/L). These results indicate that AC blending with Ni powder can improve hydrogen production in MECs while minimizing the amount of Ni in the cathode.     

Continuous H2 production during fermentation of the carbon components of lignocellulose hydrolysates: Insight into the influence of pH conditions on the conversion efficiency of individual sugars

Sugars released from lignocellulose biomass are a promising substrate for biohydrogen production. This study evaluates the effect of pH controlled between 4.0 and 7.5 on continuous dark-fermentative H₂ production from the mixture of cellobiose, xylose and arabinose. High hydrogen production rate was obtained for pH values between 6.0 and 7.0 with a maximum of 7.41 ± 0.16 L/L-d at pH 7.0. On the other hand, the highest H₂ yields of around 1.74 ± 0.02 mol/mol_consumed were obtained at pH 4.5, 5.0 and 6.0. Cellobiose was completely utilized in nearly the entire pH range, while the highest consumption of xylose and arabinose was obtained at pH 6.0 and 7.0, respectively. It shows the challenges in selecting optimum pH for fermentation of mixed sugars. Significant impact of pH conditions on the microbial structure was observed. Between pH 4.0 and 7.0 Clostridium genus dominated the consortium, while above pH 7.0 relative abundance of Bacillus genus increased significantly.     

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.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Characteristics of biohydrogen fermentation from various substrates

The characteristics of biohydrogen production from sucrose, slurry-type piggery waste and food waste under the effects of the reactor configurations and operational pHs (6 and 9) were examined by using heat-treated anaerobic sludge as a seed biomass. When sucrose was used in the batch test, the maximum hydrogen yield was 0.12–0.13 g COD (as H₂)/g COD (1.41–1.43 mol/mol hexose) at pH 6. In contrast, 0.10–0.11 g COD (as H₂)/g COD (1.12–1.21 mol/mol hexose) hydrogen yield was achieved from the reactor at pH 9. On the other hand, hydrogen production was not observed in the continuous sequencing batch mode fermenters fed with sucrose. Profile analysis at each cycle revealed hydrogen production at the initial operation periods but eventually only methane at 36 days. When slurry-type piggery waste was used as the substrate, the upflow elutriation-type fermenters produced methane but not hydrogen after 30 days operation. The fermentation intermediate profile showed that the hydrogen produced might have been consumed by homoacetogenic or propionate producing reactions, and eventually converted into methane by acetoclastic methanogens. The downflow leaching bed fermenters using food waste produced 0.013 L H₂/g volatile solids (VS) (0.0061 g COD (as H₂)/g COD) at pH 6 with 54% VS reduction whereas 0.0041 L H₂/g VS (0.0020 g COD (as H₂)/g COD) was produced at pH 9 with 86% VS reduction. The results show that the hydrogen produced should be released rapidly from the reactor before it can be consumed in other biochemical reactions, and substrates with high pH level (>9.0) can be used directly to produce hydrogen without needing to adjust the pH.