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.     

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.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

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

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

An integrated system for hydrogen and methane production during landfill leachate treatment

The patent-pending integrated waste-to-energy system comprises both a novel biohydrogen reactor with a gravity settler (Biohydrogenator), followed by a second stage conventional anaerobic digester for the production of methane gas. This chemical-free process has been tested with a synthetic wastewater/leachate solution, and was operated at 37 °C for 45 d. The biohydrogenator (system (A), stage 1) steadily produced hydrogen with no methane during the experimental period. The maximum hydrogen yield was 400 mL H₂/g glucose with an average of 345 mL H₂/g glucose, as compared to 141 and 118 mL H₂/g glucose for two consecutive runs done in parallel using a conventional continuously stirred tank reactor (CSTR, System (B)). Decoupling of the solids retention time (SRT) from the hydraulic retention time (HRT) using the gravity settler showed a marked improvement in performance, with the maximum and average hydrogen production rates in system (A) of 22 and 19 L H₂/d, as compared with 2–7 L H₂/d in the CSTR resulting in a maximum yield of 2.8 mol H₂/mol glucose much higher than the 1.1–1.3 mol H₂/mol glucose observed in the CSTR. Furthermore, while the CSTR collapsed in 10–15 d due to biomass washout, the biohydrogenator continued stable operation for the 45 d reported here and beyond. The methane yield for the second stage in system (A) approached a maximum value of 426 mL CH₄/gCOD removed, while an overall chemical oxygen demand (COD) removal efficiency of 94% was achieved in system (A).     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

Performance of a groove-type photobioreactor for hydrogen production by immobilized photosynthetic bacteria

The biofilm technique has been proved to be an effective cell immobilization method for wastewater biodegradation but it has had restricted use in the field of photobiological H₂ production. In the present study, a groove-type photobioreactor was developed and it was shown that a groove structure with large specific surface area was beneficial to cell immobilization and biofilm formation of the photosynthetic bacteria on photobioreactor surface as well as light penetration. A series of experiments was carried out on continuous hydrogen production in the groove-type photobioreactor illuminated by monochromatic LED lights and the performance was investigated. The effects of light wavelength, light intensity, inlet glucose concentration, flow rate and initial substrate pH were studied and the results were compared with those obtained in a flat panel photobioreactor. The experimental results show that the optimum operational conditions for hydrogen production in the groove-type photobioreactor were: inlet glucose concentration 10 g/L, flow rate 60 mL/h, light intensity 6.75 W/m², light wavelength 590 nm and initial substrate pH 7.0. The maximum hydrogen production rate, H₂ yield and light conversion efficiency in the groove-type photobioreactor were 3.816 mmol/m²/h, 0.75 molH₂/molglucose and 3.8%, respectively, which were about 75% higher than those in the flat panel photobioreactor.     

Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

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.     

Enhancement of biohydrogen producing using ultrasonication

Ultrasonication was evaluated as a pretreatment for biological hydrogen production from glucose in batch studies, in comparison with heat-shock pretreatment, acid pretreatment, and base pretreatment. The optimized sonication energy for hydrogen production using anaerobic digester sludge was 79 kJ/gTS. Sonication with temperature control (less than 30 °C) increased volumetric hydrogen production by 120% over the untreated sludge, and by 40% over the heat-shock and acid pretreated sludge, with a marginal (∼10%) increase in hydrogen production rate. Upon comparing the molar hydrogen yield in sonicated sludge with and without temperature control, the deleterious effect of heat on some hydrogen producers as reflected by a 30% decrease in yield to 1.03 mol H₂/mol glucose is evident. Sonication with temperature control affected a 45% increase in molar hydrogen yield to 1.55 mol H₂/mol glucose over heat-shock pretreatment at 70 °C for 30 min and acidification to pH 3.0 for 24 h at 4 °C. Sonication with temperature control produced a biomass yield of 0.13 g VSS/g COD, as compared to 0.24 g VSS/g COD for the untreated sludge. The hydrogen yield increased linearly with the molar acetate to butyrate ratio and decreased linearly with the biomass yield.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

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.     

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.     

Biohydrogen production from purified terephthalic acid (PTA) processing wastewater by anaerobic fermentation using mixed microbial communities

Purified terephthalic acid (PTA) processing wastewater was evaluated as a fermentable substrate for hydrogen (H₂) production with simultaneous wastewater treatment by dark-fermentation process in a continuous stirred-tank reactor (CSTR) with selectively enriched acidogenic mixed consortia under continuous flow condition in this paper. The inoculated sludge used in the reactor was excess sludge taken from a second settling tank in a local wastewater treatment plant. Under the conditions of the inoculants not less than 6.3 gVSS/L, the organic loading rate (OLR) of 16 kgCOD/m³ d, hydraulic retention time (HRT) of 6 h and temperature of (35 ± 1) °C, when the pH value, alkalinity and oxidation–reduction potential (ORP) of the effluent ranged from 4.2 to 4.4, 280 to 350 mg CaCO₃/L, and −220 to −250 mV respectively, soluble metabolites were predominated by acetate and ethanol, with smaller quantities of propionate, butyrate and valerate. Stable ethanol-type fermentation was formed with the sum of ethanol and acetate concentration ratio of 70.31% to the total liquid products after 25 days operation. The H₂ volume content was estimated to be 48–53% of the total biogas and the biogas was free of methane throughout the study. The average biomass concentration was estimated to be 10.82 gVSS/L, which favored H₂ production efficiently. The rate of chemical oxygen demand (COD) removal reached at about 45% and a specific H₂ production rate achieved 0.073 L/gMLVSS d in the study. This CSTR system showed a promising high-efficient bioprocess for H₂ production from high-strength chemical wastewater.     

Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

Bioconversion of de-oiled Jatropha Waste (DJW) to hydrogen and methane gas by anaerobic fermentation: Influence of substrate concentration,temperature and pH

Cellulosic materials-based de-oiled Jatropha Waste (DJW) was fermented to H₂ and CH₄ using sewage sludge inoculum. Batch assays were performed at various substrate concentrations (40–240 g/L), temperatures (25–65 °C) and pHs (5.5–7.5). The peak hydrogen production rate (HPR) and hydrogen yield (HY) of 744.0 ± 11.3 mL H₂/L-d and 10.6 ± 0.2 mL H₂/g VS obtained when the optimal substrate concentration, pH, temperature were 200 g/L, 6.5, 55 °C, respectively. The peak methane production rate (MPR) of 178.4 ± 5.6 mL CH₄/L-d obtained while DJW concentration, pH, temperature were 200 g/L, 7.0, 45 °C, however, peak methane yield (MY) of 23.3 ± 0.1 mL CH₄/g VS obtained at 40 g/L, 7.0 and 55 °C, respectively. Effect of substrate concentration on HPR and MPR was elucidated using Monod model. Butyrate and acetate were the main soluble metabolic products. Maximal carbohydrate removal and COD reduction were achieved as 51.7 ± 0.7% and 68.3 ± 1.6%, respectively.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Sequencing batch reactor enhances bacterial hydrolysis of starch promoting continuous bio-hydrogen production from starch feedstock

Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H₂ fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H₂ from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 °C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H₂ production with the starch hydrolysate, the highest H₂ production rate and yield was 0.52 L/h/L and 13.2 mmol H₂/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH₄HCO₃) concentration was 2.62 g/L, attaining a good H₂ production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H₂ fermentation while using dark fermentation effluents as the substrate.