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.     

Comparative evaluation of the mesophilic and thermophilic biohydrogen production at optimized conditions using tequila vinasses as substrate

The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H₂) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 °C and 55 °C, and pH of 5.5 maximized the bio-H₂ production rates, 25.5 ± 0.01 NmL h⁻¹ and 169.9 ± 8.9 NmL h⁻¹ in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H₂ production rate of 519 ± 13 NmL-H₂ L⁻¹ d⁻¹ equivalent to 750 ± 19 NmL-H₂ L_vinasse⁻¹, while the mesophilic one 448 ± 23 NmL-H₂ L⁻¹ d⁻¹ and 647 ± 33 NmL-H₂ L_vinasse⁻¹, respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H₂ production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed.     

Integrating waste activated sludge (WAS) acidification with denitrification by adding nitrite (NO2−)

Adding nitrite (NO₂⁻) to waste activated sludge (WAS) fermentation systems is an efficient approach to integrate fermentation with denitrification, and utilize volatile fatty acids (VFAs) to achieve a high nitrogen removal rate even at low C/N ratios. In this study, the effect of nitrite on the integrated WAS fermentation and denitrification (termed as WASFD) was investigated under acidic and alkaline conditions. The results indicated that adding nitrite achieved a most reduction of nitrite to N₂ and improved the acidification of WAS with high VFAs production. Under acidic condition (pH = 5), the maximum VFAs produced with nitrite addition was 3.3 times that without nitrite addition. Higher concentration of free nitrous acid (FNA) at the pH of 5 increased WAS particulates, improved the hydrolysis of organic substrates, and finally promoted VFAs yields. Under alkaline condition (pH = 9), adding nitrite only increased the VFAs production by 1.5 times, indicating that acidic condition was preferable for acidification than alkaline condition.     

Controlled oxidation-reduction potential on dark fermentative hydrogen production from glycerol: Impacts on metabolic pathways and microbial diversity of an acidogenic sludge

The oxidation-reduction potential (ORP) is an important factor in H₂ production via dark fermentation however its effect over microbial diversity in an acidogenic sludge has not, been well studied. This work studies the effect of ORP controlled by hydrogen peroxide and potassium ferricyanide on continuous hydrogen production and microbial diversity in an acidogenic sludge fed (HRT 12 h and pH 5.5) with glycerol. Results show that the more oxidizing ORP control environment (?540 mV) improves H₂ yield by 50–70% (0.31–0.51 molH₂/mol glycerol) over non-ORP control conditions. Oxidizing ORP values were shown to enrich microorganisms of the genus Clostridium, which have been linked to high H₂ yields. Therefore, controlling ORP in an acidogenic sludge was shown to directly modify microbial diversity at the genus level, and could likely to indirectly regulate metabolic function. Additionally, metabolic pathways were regulated by the kind of agent used.     

Enhanced biohydrogen production from tofu residue by acid/base pretreatment and sewage sludge addition

Anaerobic dark fermentation is considered a promising technology for clean energy production and waste reduction. In the present work, tofu residue and sewage sludge were utilized as substrates for fermentative hydrogen production. To increase the biodegradability, tofu residue was pretreated for 30 min in the presence of HCl and NaOH at various concentrations (0, 0.5, 1.0, and 2.0%), and then fermented by a thermophilic (60 °C) mixed culture. The solubility (SCOD/TCOD ratio) of the tofu residue increased from 4 to 30–40% after pretreatment, and the increased soluble constituents were mainly protein rather than carbohydrate compounds. However, in spite of a slight increase of carbohydrate solubility, the H₂ production performance was significantly enhanced by pretreatment, owing to the degradability of thermophilic cultures used on insoluble tofu residues. The limited H₂ yield of 0.30 mol H₂/mol hexose_added achieved in the raw tofu residue was increased 1.6- to 4-fold with the highest H₂ yield of 1.25 mol H₂/mol hexose_added at 1.0% HCl concentration. Carbohydrate degradation and the H₂ production rate also increased from 39 to 50–65% and 27 to 50–120 mL H₂/L/h, respectively. The role of pretreatment was not only to increase the biodegradability but also to suppress the activity of indigenous non H₂-producers such as lactic acid bacteria and propionic acid bacteria. When sewage sludge was added to acid pretreated (1.0% HCl) tofu residue as a co-substrate, the H₂ yield and H₂ production rate increased to 1.48 mol H₂/mol hexose_added and 161 mL H₂/L/h, respectively, which was attributed to the abundant minerals, vitamins, and metals contained in sewage sludge.     

Dark fermentative biohydrogen production by Acinetobacter junii-AH4 utilizing various industry wastewaters

Hydrogen (H₂) is one of the most promising renewable energy sources, anaerobic bacterial H₂ fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H₂ is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H₂ production was achieved by using the bacteria Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H₂ production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H₂ and Acinetobacter junii-AH4 has shown the highest cumulative H₂ of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H₂/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H₂ production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H₂.     

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.     

Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated Thermoanaerobacterium thermosaccharolyticum SP-H2

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55–60 °C and pH 7.5. The H₂-producing activity of the bacterium was studied using mono-, di- and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H₂ yield was observed during dark fermentation (DF) of maltose (1.91 mol H₂/mol hexose or 77.8 mmol H₂/L). The maximum H₂ production rate was observed during DF of xylose (13.3 ml H₂/g COD/h) and cellobiose (2.47 mmol H₂/L/h). The main soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H₂ content in biogas and the specific H₂ yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H₂ production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H₂ yield (152 ml H₂/g COD) and H₂ production rate (0.57 mmol H₂/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311 mg COD/L, respectively. At the same time, SP-H2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions.     

Structural optimization of biohydrogen production: Impact of pretreatments on volatile fatty acids and biogas parameters

The present study aims to describe an innovative approach that enables the system to achieve high yielding for biohydrogen (bio-H₂) production using xylose as a by-product of lignocellulosic biomass processing. A hybrid optimization technique, structural modelling, desirability analysis, and genetic algorithm could determine the optimum input factors to maximize useful biogas parameters, especially bio-H₂ and CH₄. As found, the input factors (pretreatment, digestion time and biogas relative pressure) and volatile fatty acids (acetic acid, propionic acid and butyric acid) had significantly impacted the biogas parameters and desirability score. The pretreatment factor had the most directly effect on bio-H₂ and CH₄ production among the factors, and the digestion time had the most indirectly effect. The optimization method showed that the best pretreatment was acidic pretreatment, digestion time > 20 h, biogas relative pressure in a range of 300–800 mbar, acetic acid in a range of 90–200 mg/L, propionic acid in a range of 20–150 mg/L, and butyric acid in a range of 250–420 mg/L. These values caused to produce H₂ > 10.2 mmol/L, CH₄ > 3.9 mmol/L, N₂ < 15.3 mmol/L, CO₂ < 19.5 mmol/L, total biogas > 0.31 L, produced biogas > 0.10 L, and accumulated biogas > 0.41 L.     

Preparation of active hydrogen-producing cultures from palm oil mill sludge for biohydrogen production system

Methods are investigated to prepare active hydrogen (H₂)-producing cultures originating from palm oil mill sludge using dark fermentation. The first successful method that produces potent H₂-producing cultures and avoids growing H₂-consuming methanogens involves heat pretreatment of the sludge at 100 °C for 2 h and then the sludge sample is shocked in an ice bath for 15 min. Subsequently, a glucose solution rich in nutrients (glucose-based substrate) of 14.80 g chemical oxygen demand (COD)/L is fed in to enrich the H₂-producing cultures. The H₂ production reaches 78.63% on day 31. The second method involves acid pretreatment of sludge with 10 M hydrochloric acid at pH 3 for 48 h. Glucose-based substrate of 25.47 g COD/L is fed into the system. The H₂ production is 69.41% on day 27. For both methods, the H₂ production is stable after the H₂ content reached its maximum. The operation is performed semi-continuously using a hydraulic retention time of 1 day and at 30 °C. The optimum bacterial cells-to-COD level of substrate is approximately 0.60 in the start-up medium. The fermentation medium has an optimum initial pH of 5 and a final pH of 5.2–5.3. These two methods are recommended to produce active H₂-producing cultures for plant start-up in bio-H₂ production.     

Bio-hydrogen production from cheese whey powder (CWP) solution: Comparison of thermophilic and mesophilic dark fermentations

Cheese whey powder (CWP) solution was used as the raw material for hydrogen gas production by mesophilic (35 °C) and thermophilic (55 °C) dark fermentations at constant initial total sugar and bacteria concentrations. Thermophilic fermentation yielded higher cumulative hydrogen formation (CHF = 171 mL), higher hydrogen yield (111 mL H₂ g⁻¹ total sugar), and higher hydrogen formation rate (3.46 mL H₂ L⁻¹ h⁻¹) as compared to mesophilic fermentation. CHF in both cases were correlated with the Gompertz equation and the constants were determined. Despite the longer lag phase, thermophilic fermentation yielded higher specific H₂ formation rate (2.10 mL H₂ g⁻¹cells h⁻¹). Favorable results obtained in thermophilic fermentation were probably due to elimination of H₂ consuming bacteria at high temperatures and selection of fast hydrogen gas producers.     

Effect of low digestate recirculation ratio on biofuel and bioenergy recovery in a two-stage anaerobic digestion process

A relatively high (0.2–4.3) digestate recirculation ratio (RR) is typically adopted to raise the pH and provide the dark fermentation reactor (DF) with alkalinity and hydrogen-producing microorganisms in a two-stage anaerobic digestion process. This study examined the production of bio-H₂ and bio-CH₄ from readily biodegradable organic waste in a large scale recirculated two-stage thermophilic anaerobic system to determine the effect of low RR on biofuel and bioenergy recovery. The performance of the two-stage system was evaluated at 2 hydraulic retention times (HRT) (1.1 and 2.5 d) in DF and 4 RR (0, 0.11, 0.18 and 0.25). The pH in DF was not controlled and ranged from 3.8 to 4.2. Hydrogen yield was negatively affected by digestate recirculation, while CH₄ yield, as well as H₂ and CH₄ production rates, first tended to increase and then decrease with increasing RR. Overall, biofuel and bioenergy were best recovered at an RR of 0.11, namely 1.48 L H₂/L/d, 0.88 L CH₄/L/d, 106.2 mL H₂/g VS_init.,161.3 mL CH₄/g VS_init., 7.7 kJ/g VS_init. and 88.2 kJ/L/d were obtained depending on HRT in DF. It has been shown that a low RR can improve the performance of the two-stage anaerobic digestion process.     

Mesophilic biohydrogen production from calcium hydroxide treated wheat straw

The use of Ca(OH)₂ pre-treatment to improve fermentative biohydrogen yields, from wheat straw was investigated. Wheat straw was pre-treated with 7.4% (w/w) Ca(OH)₂ at ambient temperature (20 °C) for 2, 5, 8, and 12 days, prior to 35 °C fermentation with sewage sludge inoculum. Biohydrogen yields were evaluated during dark fermentation and simultaneous saccharification fermentation (SSF) of total pre-treated straw material and compared to those from separated solid and hydrolysate fractions. Ca(OH)₂ pre-treatment followed by SSF, exhibited a synergetic relationship. On average, 58.78 mL-H₂ g-VS⁻¹ was produced from SSF of pre-treated and filtered solids. This was accompanied by approximately a 10-fold increase in volatile fatty acid production, compared to the untreated control. By omitting pre-treatment hydrolysate liquors from SSF, H₂ production increased on average by 35.8%, per VS of harvested straw. Additional inhibition studies indicated that CaCO₃, formed as a result of pre-treatment pH control, could promote homoacetogenesis and reduce biohydrogen yields.     

Enhanced methane fermentation of municipal sewage sludge by microbial electrochemical systems integrated with anaerobic digestion

Sewage sludge from a municipal wastewater treatment plant was fed into a microbial electrochemical system, combined with an anaerobic digester (MES-AD), for enhanced methane production and sludge stabilization. The effect of thermally pretreating the sewage sludge on MES-AD performance was investigated. These results were compared to those obtained from control operations, in which the sludge was not pretreated or MES integration was absent. The soluble chemical oxygen demand (SCOD) in the raw sewage sludge after pretreatment was 31% higher than the SCOD in untreated sludge (5804.85 mg/L vs. 4441.46 mg/mL). The methane yield and proportion of methane in biogas generated by the MES-AD were higher than those of the control systems, regardless of the pretreatment process. The maximum methane yield (0.28 L CH₄/g COD) and methane production (1139 mL) were obtained with the MES inoculated with pretreated sewage sludge. Methane yield and production with this system using pretreated sewage were 47% and 56% higher, respectively, than those of the control (0.19 L CH₄/g COD, 730 mL). Additionally, the maximum SCOD removal (89%) and current generation were obtained with the MES inoculated with a pretreated substrate. These results suggested that sewage sludge could be efficiently stabilized with enhanced methane production by synergistic combination of MES-AD system with pretreatment process.     

Improving bio-H2 production by manganese doped magnetic carbon

Manganese doped magnetic carbon (MDMC) was synthesized to enhance bio-hydrogen (H₂) process under the mesophilic and thermophilic conditions. MDMC characterization showed that MnFe₂O₄, Fe₂O₃ and few MnCO₃ were adhered to activated carbon and formed MDMC whose specific surface area and average pore size were 110.92 m²·g⁻¹ and 3.2 nm, respectively. Suitable concentration of MDMC promoted while high dosage suppressed the H₂ production of glucose through anaerobic fermentation by heat-treated sludge. The highest yields of 211 and 148 ml H₂/g glucose were obtained at 400 mg/L (37 °C) and 600 mg/L MDMC (55 °C), being 55.8% and 19.3% higher than that of the control groups, respectively. Some possible mechanisms were proposed as follows: appropriate concentration of MDMC provided considerable and favorable sites for microbial colonization, released trace elements (e.g. Mn, Fe) into liquid for the microbial growth, and promoted the direct inter-species electron transfer. Moreover, MDMC could be recycled by magnetic separation after bio-H₂ process due to its magnetism.     

Enhanced H2 production from corn stalk by integrating dark fermentation and single chamber microbial electrolysis cells with double anode arrangement

The low conversion efficiency of substrate is one of the main bottlenecks in dark fermentation for bio-H₂ production. Herein, an enhanced H₂ yield from corn stalk was achieved by integrating dark fermentation and single chamber microbial electrolysis cells (MECs). In the dark fermentation stage, a H₂ yield of 129.8 mL H₂/g-corn stalk and an average H₂ production rate of 1.73 m³/m³ d were recorded at 20 g/L of corn stalk and initial pH 7.0. The effluent from dark fermentation was diluted and further employed as feedstock to generate H₂ by MECs. A H₂ yield of 257.3 mL H₂/g-corn stalk, an HPR of 3.43 ± 0.12 m³/m³ d and an energy efficiency of 166 ± 10% were obtained with the effluent COD of 3995.5 mg/L under 0.8 V applied voltage. During MECs operation stage, about 90 ± 2% of acetate was converted to H₂ and the corresponding COD removal reached 44 ± 2% in MECs. Overall, the H₂ yield can reach 387.1 mL H₂/g-corn stalk by integrating dark fermentation and MECs, which had nearly tripled as against that of dark fermentation.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

Characterization of microbial community in the two-stage process for hydrogen and methane production from food waste

The structure of a microbial community in the two-stage process for H₂ and CH₄ production from food waste was investigated by a molecular biological approach. The process was a continuous combined thermophilic acidogenic hydrogenesis and mesophilic (RUN1) or thermophilic (RUN2) methanogenesis with recirculation of the digested sludge. A two-phase process suggested in this study effectively separate H₂-producing bacteria from methanogenic archaea by optimization of design parameters such as pH, hydraulic retention time (HRT) and temperature. Galore microbial diversity was found in the thermophilic acidogenic hydrogenesis, Clostridium sp. strain Z6 and Thermoanaerobacterium thermosaccharolyticum were considered to be the dominant thermophilic H₂-producing bacteria. The hydrogenotrophic methanogens were inhibited in thermophilic methanogenesis, whereas archaeal rDNAs were higher in the thermophilic methanogenesis than those in mesophilic methanogenesis. The yields of H₂ and CH₄ were in equal range depending on the characteristics of food waste, whereas effluent water quality indicators were different obviously in RUN1 and RUN2. The results indicated that hydrolysis and removal of food waste were higher in RUN2 than RUN1.     

Biogas production from sewage sludge and microalgae co-digestion under mesophilic and thermophilic conditions

Isochrysis galbana and Selenastrum capricornutum, marine and freshwater microalgae species respectively, were co-digested with sewage sludge under mesophilic and thermophilic conditions. The substrates and the temperatures significantly influenced biogas production.Under mesophilic conditions, the sewage sludge digestion produced 451 ± 12 mL_Biogas/g_SV. Furthermore, all digesters were fed with I. galbana, or mixed with sludge, resulting in an average of 440 ± 25 mL_Biogas/g_SV. On the contrary, S. capricornutum produced 271 ± 6 mL_Biogas/g_SV and in the mixtures containing sludge produced intermediate values between sludge and microalgae production.Under thermophilic conditions, the sewage sludge digestion achieved yet the highest biogas yield, 566 ± 5 mL_Biogas/g_SV. During co-digestion, biogas production decreased when the microalgae content increased, and for I. galbana and for S. capricornutum it reached minimum values, 261 ± 11 and 185 ± 7 mL_Biogas/g_SV, respectively. However, no evidence of inhibition was found and the low yields were attributed to microalgae species characteristics.The methane content in biogas showed similar values, independently from the digested substrate, although this increased by approximately 5% under thermophilic condition.     

Effects of starch loading rate on performance of combined fed-batch fermentation of ground wheat for bio-hydrogen production

Ground wheat powder solution (10 g L⁻¹) was subjected to combined dark and light fermentations for bio-hydrogen production by fed-batch operation. A mixture of heat treated anaerobic sludge (AN) and Rhodobacter sphaeroides-NRRL (RS-NRRL) were used as the mixed culture of dark and light fermentation bacteria with an initial dark/light biomass ratio of 1/2. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. The highest cumulative hydrogen formation (CHF = 3460 ml), hydrogen yield (201 ml H₂ g⁻¹ starch) and formation rate (18.1 ml h⁻¹) were obtained with a starch loading rate of 80.4 mg S h⁻¹. Complete starch hydrolysis and glucose fermentation were achieved within 96 h of fed-batch operation producing volatile fatty acids (VFA) and H₂. Fermentation of VFAs by photo-fermentation for bio-hydrogen production was most effective at starch loading rate of 80.4 mg S h⁻¹. Hydrogen formation by combined fermentation took place by a fast dark fermentation followed by a rather slow light fermentation after a lag period.