BioH2 production from food waste by anaerobic membrane bioreactor

In this study, long term continuous bioH₂ and volatile fatty acids (VFA) production potential in anaerobic membrane bioreactors (anMBR) from food waste by dark fermentation is investigated. Experiments of total 868 days are divided into 8 periods, each reflecting different operating conditions such as organic loading rates (OLR), hydraulic retention times (HRT) and pH regimes. Taking advantage of membrane technology, reactors are operated at solids content high as 50 g L^_1 and reached to average 147 mL H₂/g VS added production potential. The most effective H₂ production, coupled with highest hydrolysis and acidification efficiency is achieved with pH = 7, 5 days of HRT and 18 kg COD m⁻³ d⁻¹ of organic loading rate. It is also observed that in the periods which highest bioH₂ production was achieved, were also the periods with highest B/A ratios. Microbial community was dominated by butryic acid producers according to Denaturing Gradient Gel Electrophoresis (DGGE) and clonning analysis and it is concluded that butyric acid pathway is the dominant bioH₂ production mechanism in this study. With the membrane-supported completeley stirred tank reactor system, suitable conditions are not only provided for high bioH₂ production, but also for the production of solids free permeate with volatile fatty acids content high as 29,765 mg L⁻¹. Thus, with the innovative system proposed in this study, it is possible to establish a plant model suitable for the biorefinery concept.     

Strategies to improve the biohydrogen production from cassava wastewater in fixed-bed reactors

The biological production of hydrogen from cassava starch wastewater (CSW) was evaluated in an anaerobic fixed-bed reactor (UAFBR). The assays were carried out to evaluate the effects of organic loading rate (OLR) increase and strategies of inoculation (AS – anaerobic sludge thermally treated and NF – naturally fermented cassava starch wastewater) on UAFBR performance. The OLR increase (10–20 g L⁻¹ d⁻¹) associated with hydraulic retention time (HRT) decrease (4–2 h) improved the volumetric hydrogen production rate (VHPR, from 229 to 550 mLH₂.L⁻¹.d⁻¹), molar hydrogen flow rate (MHFR, from 1.0 to 2.5 mmolH₂.h⁻¹) and hydrogen yield (HY, from 0.2 to 0.3 molH₂.mol⁻¹Carb) from CSW due to increase in substrate availability. Both inoculation alternatives (AS and NF) were effective for the selection of acidogenic microorganisms, which demonstrates that NF could be considered a simple and economic alternative for the acquisition of inoculum for continuous acidogenic reactors. Hydrogen production decreased after 10 days of operation when the specific organic loading rate (SOLR) reached reduced values (<1 gCarb.g⁻¹VSS.d⁻¹), which impairs hydrogen production. For all assays, methane was present in the biogas after the 20th day of operation mainly due to biomass accumulation, which alters the biota of the reactor. Although many factors could influence the process performance in UAFBR for the production of biohydrogen, the accumulation of biomass have been pointed as the main factor in the determination of the production time, thus demanding the implementation of systematic practices to remove the excess of biomass to maintain the SOLR in levels adequate for hydrogen production.     

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.     

Thermophilic biohydrogen production from sugarcane molasses under low pH: Metabolic and microbial aspects

By-products from sugarcane mills have a considerable energy potential, and therefore have been studied aiming to generate biogas emphasising biohydrogen (bioH₂). Sugarcane molasses, a byproduct from sugar production, are rich in carbohydrates, thus easily biodegraded by anaerobic microorganisms. This study evaluated the production of bioH₂ in unfavorable pH (3.80) using molasses as a feedstock in an anaerobic structured bed reactor (AnSTBR-A) under thermophilic conditions (55 °C). The AnSTBR-A operated with an organic loading rate (OLR) of 60 g L⁻¹ d⁻¹ was able to produce bioH₂ under long-term operation (392 days). The hydrogen yield (HY) was 1.18 mol H₂ mol total carbohydrates⁻¹. The results highlighted HY variation concomitant with metabolite concentrations. The main role to bioH₂ production in AnSTBR-A was acetate + lactate → butyric + bioH₂, with a predominance of the organism belonging to the Thermoanaerobacterium genus.     

Enhanced hydrogen production by a newly described heterotrophic marine bacterium, Vibrio tritonius strain AM2, using seaweed as the feedstock

To achieve more stable bio-hydrogen (bioH₂) production from non-food feedstocks, stable feedstock preparations of marine biomass and an efficient bioH₂ system using marine bacteria under saline conditions are two important key technologies that needed to be developed. Vibrio tritonius strain AM2, which was isolated from the gut of a marine invertebrate, was cultured under various conditions in marine broth (at initial 2.25% (w/v) NaCl) supplemented with mannitol, a seaweed carbohydrate, to evaluate its hydrogen production. The maximum molar yield of bioH₂ was recorded as 1.7 mol H₂/mol mannitol at pH 6 and 37 °C. The mannitol-grown cells had higher yields of bioH₂ than the glucose-grown cells in the pH range 5.5–7.5. Compared to glucose, mannitol might be a better substrate for bioH₂ production using strain AM2. Fermentation product profiling revealed that strain AM2 might be utilising the formate-hydrogen pathway for bioH₂ production. Furthermore, strain AM2 was able to produce hydrogen from powdered brown macroalgae containing 31.1% dry weight of mannitol. The molar yield of hydrogen reached 1.6 mol H₂/mol mannitol contained in the seaweed feedstock. In conclusion, strain AM2 has the ability to produce hydrogen from mannitol with high yields even under saline conditions.     

The production of electricity from dual-chambered microbial fuel cell fueled by old age leachate

In this study, electricity production from old age landfill leachate was investigated using dual chambered microbial fuel cell with Ti-TiO₂ electrodes. The effect of organic loading rate on microbial fuel cell performance was examined by changing the hydraulic retention time and leachate chemical oxygen demand (COD) concentration. Microbial diversity at different conditions was studied using PCR-DGGE profiling of 16 sRNA fragments of microorganisms in the liquid media of the anode chamber and of the biofilm on the anode electrode. Both COD removal and current density were positively affected with increasing organic loading rate. The highest microbial fuel cell performance was achieved at hydraulic retention time of 0.5 day and organic loading rate of 10 g COD/L.day. The performance of the microbial fuel cell reactor decreased when hydraulic retention time was reduced to 0.25 day. The investigation of the microbial dynamics indicated that abundance of bacterial species was considerably dependent on the operational conditions. The microbial fuel cell reactor was mainly dominated by Geobacter, Shewanella, and Clostridium species, and some bacteria were easily washed out at lower hydraulic retention times.     

Extreme thermophilic condition: An alternative for long-term biohydrogen production from sugarcane vinasse

Boosted by the high temperatures in which vinasse is generated (90 °C–100 °C), this study evaluated the effect of an extreme thermophilic condition (70 °C) on sugarcane vinasse Dark Fermentation (DF) in an Anaerobic Structured Bed Reactor (ASTBR). Four hydraulic retention times (HRT) (19, 15, 12 and 8 h) were evaluated. Higher HRT resulted in a greater H₂ production rate (690 mLH₂.d⁻¹.L⁻¹), higher yields (1.8 molH₂.mol_Glucose⁻¹) and greater stability. The extreme temperature inhibits microorganisms' extracellular polymer production, thus leading to a disperse growth, preventing excess biomass accumulation, which was previously reported as the main drawback in H₂ production at lower temperatures. The ASTBR higher void index is also responsible for lower biomass/solids retention. The H₂ production main route was through the lactic/acetic acid pathway, which is highly reliant on the pH of fermentation broth. The main genus involved in H₂ production at 70 °C were Clostridium, Pectinatus, Megasphaera and Lactobacillus.     

Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater

Production of biohydrogen using dark fermentation has received much attention owing to the fact that hydrogen can be generated from renewable organics including waste materials. The key to successful application of anaerobic fermentation is to uncouple the liquid retention time and the biomass retention time in the reactor system. Various reactor designs based on biomass retention within the reactor system have been developed. This paper presents our research work on bioreactor designs and operation for biohydrogen production. Comparisons between immobilized-cell systems and suspended-cell systems based on biomass growth in the forms of granule, biofilm and flocs were made. Reactor configurations including column- and tank-based reactors were also assessed. Experimental results indicated that formation of granules or biofilms substantially enhanced biomass retention which was found to be proportional to the hydrogen production rate. Rapid hydrogen-producing culture growth and high organic loading rate might limit the application of biofilm biohydrogen production, since excessive growth of fermentative biomass would result in washout of support carrier. It follows that column-based granular sludge process is a preferred choice of process for continuous biohydrogen production from organic wastewater, indicating maximum hydrogen yield of 1.7 mol-H₂/mol-glucose and hydrogen production rate of 6.8 L-H₂/L-reactor h.     

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH₂) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH₂ production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L⁻¹ bromoethanesulphonic acid, 24 h) on bioH₂ production from starch powder (1% w/v) showed highest yield (323.4 mL g⁻¹ starch) in heat-treatment and peak production rate (144.5 mL L⁻¹ h⁻¹) in acid-treatment. Acetate (1.07 g L⁻¹) and butyrate (1.21 g L⁻¹) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH₂ production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H₂ yielding conditions. Peak H₂ yield and production rate were 412.6 mL g⁻¹ starch and 78.6 mL L⁻¹ h⁻¹, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH₂ yield of 126.5 mL g⁻¹ COD and 456 mL g⁻¹ starch. The net energy was calculated to be +2.97 kJ g⁻¹ COD and +0.57 kJ g⁻¹ COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 10¹³ kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH₂ production with low energy input.     

Biomethanation potential of marine macroalgal Ulva biomass in sequencing batch mode: Changes in process performance and microbial community structure over five cycles

Anaerobic digestion (AD) of Ulva biomass, a promising next-generation feedstock for energy production, was investigated in sequencing batch mode. Over five cycles of operation, the methane yield decreased more than twofold (from 0.15 to 0.07 L/g COD_fed), while the organic treatment efficiency (i.e., chemical oxygen demand (COD) removal) remained fairly constant (53.7–61.1%). Such changes in reactor performance were related with structural variations in the microbial community, particularly the bacterial community, with repeated cycles. Methanosaeta- and Methanolinea-related populations were most likely the main aceticlastic and hydrogenotrophic methanogens, respectively, in the reactor. The emergence and prevalence of sulfate-reducing bacteria (SRBs), primarily a Solitalea-related population, most likely resulted in increased consumption of organic substrates for sulfate reduction, rather than methane production, in later cycles. Our observations suggest that the metabolic properties of the reactor changed with the transition of the bacterial community structure over cycles, and the metabolic shift had a negative effect on methanogenesis. The sequencing batch operation strategy applied in this study was not suitable for maximizing methane production from Ulva biomass, although the treatment efficiency was fairly stable. Robust control of SRB activity is necessary for more stable and efficient biomethanation of Ulva biomass in sequencing batch mode.     

Influence of reactor configuration on fermentative hydrogen production during wastewater treatment

Influence of reactor configuration [biofilm/suspended growth] on fermentative hydrogen (H₂) production and substrate degradation was evaluated employing anaerobic mixed consortia. Reactors were operated at acidophilic (pH 6.0) condition employing designed synthetic wastewater as substrate at an organic loading rate of 3.4 Kg COD/m³-day with a retention time of 24 h at 28 ± 2 °C. Experimental data enumerated the influence of reactor configuration on both H₂ production and wastewater treatment. Biofilm reactor (28.98 mmol H₂/day; 1.25 Kg COD/m³-day) showed relatively efficient performance over the corresponding suspended growth configuration (20.93 mmol H₂/day; 1.08 Kg COD/m³-day). Specific H₂ yields of 6.96 mmol H₂/g-COD_L-day (19.32 mmol H₂/g-COD_R-day) and 5.03 mmol H₂/g-COD_L-day (16.10 mmol H₂/g-COD_R-day) were observed during stabilized phase of operation of biofilm and suspended growth reactors respectively. Higher concentration of VFA generation was observed in the biofilm reactor. Both the configurations recorded higher acetate concentration over other soluble metabolites indicating the dominance of acid-forming metabolic pathway during the H₂ production process.     

Bio-electrochemical evaluation of fermentative hydrogen production process with the function of feeding pH

Fermentative hydrogen (H₂) production process in concurrence with feeding pH [aciodophilic (pH 6.0) and neutral (pH 7.0)] and reactor operation mode (continuous and fed-batch) was evaluated in a biofilm configured reactor [upflow mode; retention time, 24 h; operating temperature, 28 ± 2 °C; organic loading rate, 3.4 kg COD/m³ day] using anaerobic mixed consortia. Acidophilic pH showed relatively effective performance with respect to H₂ production compared to neutral operation. Neutral pH illustrated effective substrate removal efficiency over the corresponding acidophilic operation. Fed-batch mode of operation with acidophilic pH showed highest H₂ production among the studied experimental variations. The pattern of soluble metabolites distribution showed the persistence of acid-forming metabolic flow associated with acidogenesis which may be considered as optimum microenvironment for effective H₂ production. Bio-electrochemical behavior of mixed anaerobic consortia (whole cell) during H₂ production process was evaluated employing cyclic voltammetry (CV) in electrochemical cell [platinum as working electrode; Ag/AgCl as reference electrode; graphite rod as counter electrode; wastewater as electrolyte] to gain insight into the possible mechanism based on intracellular electron transfer involved in the fermentative metabolic process. Voltammogram profiles visualized well defined redox pairs in forward and reverse scans at both pH conditions and the signals corresponded to intracellular electron carrier, NADH/NAD⁺ (E^0′, −0.32 V). Relatively higher energy output was observed in acidophilic operation which might be attributed to the possibility of efficient proton (H⁺) transfer between metabolic intermediates.     

Effect of hydraulic retention time on the hydrogen production in a horizontal and vertical continuous stirred-tank reactor

Hydraulic retention time (HRT) is the main process parameter for biohydrogen production by anaerobic fermentation. This paper investigated the effect of the different HRT on the hydrogen production of the ethanol-type fermentation process in two kinds of CSTR reactors (horizontal continuous stirred-tank reactor and vertical continuous stirred-tank reactor) with molasses as a substrate. Two kinds of CSTR reactors operated with the organic loading rates (OLR) of 12kgCOD/m3•d under the initial HRT of the 8 h condition, and then OLR was adjusted as 6kgCOD/m3•d when the pH drops rapidly. The VCSTR and HCSTR have reached the stable ethanol-type fermentation process within 21 days and 24 days respectively. Among the five HRTs settled in the range of 2–8 h, the maximum hydrogen production rate of 3.7LH₂/Ld and 5.1LH₂/Ld were investigated respectively in the VCSTR and HCSTR. At that time the COD concentration and HRT were 8000 mg/L and 5 h for VCSTR, while 10000 mg/L and 4 h for HCSTR respectively.Through the analysis on the composition of the liquid fermentation product and biomass under the different HRT condition in the two kinds of CSTR, it can found that the ethanol-type fermentation process in the HCSTR is more stable than VCSTR due to enhancing biomass retention of HCSTR at the short HTR.     

Adaptation of biohydrogen producing reactor to higher substrate load: Redox controlled process integration strategy to overcome limitations

Adaptation of acidogenic sequencing batch biofilm reactor (AcSBBR) to higher loading conditions of vegetable waste extract was studied during biohydrogen production at pH 6.0 under ambient conditions. H₂ production rate (HPR) and cumulative H₂ production (CHP) were found to improve with increase in organic load from 4.50 to 26.44 kg COD/m³ and later at 35.25 kg COD/m³ stabilization was observed. Acid metabolic intermediates production tends to lower the system pH which limits the substrate degradation and H₂ production at higher loading conditions. To overcome these limitations, redox controlled strategy (pH 7.0) was applied by integrating another AcSBBR. Upon redox controlled integration, CHP and substrate degradation were found to improve by 42.81% and 36.82% respectively. This approach helped to maintain the favorable redox microenvironment for fermentation at higher VFA concentrations. This process integration methodology will help to overcome some persistent limitation observed during biohydrogen production and make the process sustainable especially with high strength waste/wastewaters.     

Hydrogen and methane production in extreme thermophilic conditions in two-stage (upflow anaerobic sludge bed) UASB reactor system

Two-stage hydrogen and methane production in extreme thermophilic (70 °C) conditions was demonstrated for the first time in UASB-reactor system. Inoculum used in hydrogen and methane reactors was granular sludge from mesophilic internal circulation reactor and was first acclimated for extreme thermophilic conditions. In hydrogen reactor, operated with hydraulic retention time (HRT) of 5 h and organic loading rate (OLR) of 25.1 kg COD/m³/d, hydrogen yield was 0.73 mol/mol glucose_added. Methane was produced in second stage from hydrogen reactor effluent. In methane reactor operated with HRT of 13 h and OLR of 7.8 kg COD/m³/d, methane yield was 117.5 ml/g COD_added. These results prove that hydrogen and methane can be produced in extreme thermophilic temperatures, but as batch experiments confirmed, for methane production lower temperature would be more efficient.     

Hydrogen production using sono-biohydrogenator

Hydrogen production in a novel sonicated biological hydrogen reactor (SBHR) was investigated and compared with a continuous stirred tank reactor (CSTR). The two systems were operated at a hydraulic retention time (HRT) of 12 h and two organic loading rates (OLRs) of 21.4 and 32.1 g COD/L.d. The average hydrogen production rates per unit reactor volume for the conventional CSTR were 2.6 and 2.8 L/L.d, as compared with 4.8 and 5.6 L/L.d for SBHR, at the two OLRs, respectively. Hydrogen yields of 1.2 and 1.0 mol H₂/mol glucose were observed for the CSTR, respectively, while for the SBHR, the hydrogen yields were 2.1 and 1.9 mol H₂/mol glucose at the two OLRs, respectively. The hydrogen content in the SBHR’s headspace was higher than that in CSTR by 10% and 31% at OLRs of 21.4 and 32.1 g COD/L.d, respectively. Both glucose conversion efficiency and HAc/HBu ratio in the SBHR were higher than in the conventional CSTR at both OLRs. The biomass yield of about 0.32 g VSS/g COD observed in the CSTR and 0.23 g VSS/g COD in the SBHR substantiate the higher H₂ yield in the SBHR. DGGE analysis confirmed the specificity of the microbial hydrogen-producing culture in the SBHR, with two different hydrogen producers (Clostridium sp. and Citrobacter freundii) detected in the SBHR and not detected in the CSTR.     

Two-stage UASB reactor converting coffee drink manufacturing wastewater to hydrogen and methane

In this study, the feasibility of a continuous two-stage up-flow anaerobic sludge blanket (UASB) reactor system, consisted of thermophilic (55 °C) dark fermentative H₂ production and mesophilic (35 °C) CH₄ production from coffee drink manufacturing wastewater (CDMW), was tested. A recently proposed operational strategy was used to overcome a major drawback of the long start-up period of the UASB reactor. Firstly, a completely stirred tank reactor (CSTR) was operated for 8 days to prepare seeding. The seed was then directly transferred to the UASB reactor. Microbial aggregation took place in the initial period, and the floc size was gradually increased over time. In UASB reactor, the maximum H₂ yield of 2.57 mol H₂/mol hexose_added and a stable H₂ production rate of 4.24 L H₂/L/h were observed at a hydraulic retention time (HRT) of 6 h and substrate concentration of 20 g Carbo. COD/L. In this novel method using CDMW, thermophilic H₂-producing granules with an average particle size of 1.3 mm was successfully developed after 100 days. The more bioenergy recovery was attempted in a post-treatment process using a mesophilic UASB reactor for CH₄ production from the H₂ fermented effluent. The maximum CH₄ yield of 325 mL of CH₄/g COD was achieved with removing 93% of the COD at an organic loading rate of 3.5 g COD/L/d. The developed two-stage UASB reactor system achieved biogas conversion by 88.2% (H₂ 15.2% and CH₄ 73%) and COD removal by 98%.     

Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation

Biohydrogen is usually produced via dark fermentation, which generates CO₂ emissions and produces soluble metabolites (e.g., volatile fatty acids) with high chemical oxygen demand (COD) as the by-products, which require further treatments. In this study, mixotrophic culture of an isolated microalga (Chlorella vulgaris ESP6) was utilized to simultaneously consume CO₂ and COD by-products from dark fermentation, converting them to valuable microalgae biomass. Light intensity and food to microorganism (F/M) ratio were adjusted to 150 μmol m⁻² s⁻¹ and F/M ratio, 4.5, respectively, to improve the efficiency of assimilating the soluble metabolites. The mixotrophic microalgae culture could reduce the CO₂ content of dark fermentation effluent from 34% to 5% with nearly 100% consumption of soluble metabolites (mainly butyrate and acetate) in 9 days. The obtained microalgal biomass was hydrolyzed with 1.5% HCl and subsequently used as the substrate for bioH₂ production with Clostridium butyricum CGS5, giving a cumulative H₂ production of 1276 ml/L, a H₂ production rate of 240 ml/L/h, and a H₂ yield of 0.94 mol/mol sugar.     

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.     

Biological hydrogen production in continuous stirred tank reactor systems with suspended and attached microbial growth

Fermentative H₂ production in continuous stirred tank reactor (CSTR) system with bacteria attached onto granular activated carbon (GAC) was designed to produce H₂ continuously. The H₂ production performances of CSTR with suspended and attached-sludge from molasses were examined and compared at various organic loading rates (8–40 g COD/L/d) at hydraulic retention time of 6 h under mesophilic conditions (35 °C). Both reactor systems achieved ethanol-type fermentation in the pH ranges 4.5–4.8 and 3.8–4.4, respectively, while ORP ranges from −450 to −470 mV and from −330 to −350 mV, respectively. The hydrogen production rate in the attached system was higher compared to that of the suspended system (9.72 and 6.65 L/d/L, respectively) while specific hydrogen production rate of 5.13 L/g VSS/d was higher in the suspended system. The attached-sludge CSTR is more stable than the suspended-sludge CSTR with regard to hydrogen production, pH, substrate utilization efficiency and metabolic products (e.g., volatile fatty acids and ethanol) during the whole test.