Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

Production of biohydrogen from waste wheat in continuously operated UPBR: The effect of influent substrate concentration

Utilization of waste materials is one of the most economical approaches to biohydrogen production. Continuous generation of biohydrogen in a bioreactor makes the process more economical with respect to the conventional physical and chemical method. The two main parameters that affect the biohydrogen production in a continuously operated bioreactor are hydraulic retention time (HRT) and influent substrate concentrations. The effect of influent substrate concentration on biohydrogen generation in an up-flow packed bed reactor (UPBR) at HRT = 3 h was investigated in this study. The substrate was waste wheat which was acid hydrolyzed in H₂SO₄ by adjusting the pH value to pH = 2, under high temperature as T = 90 °C in an autoclave to obtain fermentable sugar solution. A natural and porous support particle namely, aquarium biological sponge (ABS) was the microbial immobilization surface in the reactor. Total and hydrogen gas volumes, hydrogen percentage, influent and effluent substrate concentrations, VFA concentrations were monitored. The influent substrate concentration (TS_o) was varied between TS_o = 10 g/L and TS_o = 35 g/L. The process performance was evaluated as biohydrogen volume, percentages, rate and yield under varying operating conditions. The production volume (4275 ml/day) and the rate (3.05 L H₂/L day) were maximum at influent sugar concentration of TS_o = 25 g/L, but the yield reached to its maximum value as Y = 1.22 mol H₂/mol glucose at TS_o = 19 g/L. Substrate limitation and inhibitions were observed at influent concentrations of TS_o = 10 g/L and TS_o = 35 g/L, respectively. The results indicated that ABS could be suggested as a microbial support particle for hydrogen generation in immobilized systems.     

Continuous biohydrogen production in immobilized biofilm system versus suspended cell culture

In this study, the performance of a new cell immobilization material, namely ceramic ball, was examined for continuous biohydrogen production in comparison to suspended cell culture system (CSTR). Production of biohydrogen in both systems was assessed under thermophilic conditions. Both systems were operated at varying hydraulic retention times (HRT) by shortening HRT values from 24 to 1.5 h at an influent sucrose concentration of 10 g/l. The immobilized bioreactor configuration outcompeted the CSTR bioreactor in terms of both volumetric hydrogen production (2.7 l H₂/l/day for immobilized system @ HRT = 3 h and 0.5 l H₂/l/day for CSTR @ HRT = 24 h) and resistance to cell-washout (CSTR reactor lost significant amount of biomass at short HRT values). It was concluded that immobilized bioreactor configuration is much more robust than CSTR against high organic loading rates and 5 fold more volumetric hydrogen production was achieved in 8 fold smaller immobilized bioreactor.     

The effect of hydraulic retention time on thermophilic dark fermentative biohydrogen production in the continuously operated packed bed bioreactor

The main objective of the study is to investigate the effect of hydraulic retention times on continuous dark fermentative biohydrogen production in an up-flow packed bed reactor (UPBR) containing a novel microorganism immobilization material namely polyester fiber beads. The hydrogen producing dark fermentative microorganisms were obtained by heat-pretreatment of anaerobic sludge from the acidogenic phase of an anaerobic wastewater treatment plant. Glucose was the sole carbon source and the initial concentration was 15 ± 1 g/L throughout the continuous feeding. UPBR was operated under the thermophilic condition at T = 48 ± 2 °C and at varying HRTs between 2 h and 6 h. The hydrogen productivity of continuously operated UPBR increased with increasing HRT. Hydrogen production volume varied between 4331 and 6624 ml/d, volumetric hydrogen production rates (VHPR) were obtained as 3.09–4.73 L H₂/L day, and hydrogen production yields (HY) were 0.49 mol/mol glucose-0.89 mol/mol glucose depending on HRT. Maximum daily hydrogen volume (6624 ml/d), the yield (0.89 mol/mol glucose) and VHPR (4.73 L H₂/L day) were obtained at HRT = 6 h. The production rate and the yield decreased with increasing organic loading rate due to substrate inhibition.     

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal was investigated at different feeding intervals. Ground wheat was acid hydrolyzed at pH = 3 and T = 121 °C for 30 min using an autoclave. The resulting sugar solution was subjected to dark fermentation with periodic feeding and effluent removal. The feed solution contained 9 ± 0.5 g L⁻¹ total sugar supplemented with some nutrients. Depending on the feeding intervals hydraulic residence time (HRT) was varied between 6 and 60 h. Steady-state daily hydrogen production increased with decreasing HRT. The highest daily hydrogen production (305 ml d⁻¹) and volumetric hydrogen production rate (1220 ml H₂ L⁻¹ d⁻¹) were obtained at HRT of 6 h. Hydrogen yield (130 ml H₂ g⁻¹ total sugar) reached the highest level at HRT = 24 h. Effluent total sugar concentration decreased, biomass concentration and yield increased with increasing HRT indicating more effective sugar fermentation at high HRTs. Dark fermentation end product profile shifted from acetic to butyric acid with increasing HRT. High acetic/butyric acid ratio obtained at low HRTs resulted in high hydrogen yields.     

Comparison of thermophilic and hyperthermophilic dark fermentation with subsequent mesophilic methanogenesis in expanded granular sludge bed reactors

Herein, dark fermentation (DF, V = 5.5 L) and subsequent mesophilic methanogenesis (V = 43.5 L) are run as expanded granular sludge bed reactors (EGSB) at thermophilic (υDF = 60 °C) and hyperthermophilic (υDF = 80 °C) temperatures. A synthetic glucose wastewater is run with a 22.5 g/L chemical oxygen demand (COD) and 48–9 h hydraulic retention times (HRTs), giving organic loading rates (OLRs) of 11–60 g COD/L/d for DF. The maximum hydrogen production rate (HPR) is HPR = 3.0 m³/m³/d for HRT = 9 h with a 50 L/kg COD hydrogen yield (HY) and 40 vol% H₂. Methane production rate (MPR) reaches MPR = 2.6 m³/m³/d with 70 vol% CH₄ at HRT = 2.8 d. The highest H₂ yields are HY = 180 L/kg COD with 53 vol% H₂ (thermophilic, HRT = 48 h). Hyperthermophilic temperatures led to lower HPRs (0.7 m³/m³/d) and MPRs (1.6 m³/m³/d). 53% of Thermoanaerobacterium thermosaccharolyticum as an H₂ producer are found. Discoloration of granular sludge from black to white and granule stability was observed in DF.     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

Biohydrogen from molasses with ethanol-type fermentation: Effect of hydraulic retention time

Though ethanol-type fermentation has many advantages for improving hydrogen production rate (HPR) in continuously mode hydrogen producing system, information on this fermentation is very deficient. The effect of hydraulic retention time (HRT) on biohydrogen production and operational stability of ethanol-type fermentation was investigated in a continuous stirred tank reactor (CSTR) using molasses as substrate. Five HRTs were examined, ranging from 4 to 10 h. At HRT 5 h, the highest HPR of 12.27 mmol L⁻¹ h⁻¹ was obtained from ethanol-type fermentation in the pH range of 4.3–4.4. During the whole operation process, ethanol, butyrate and acetate were the predominant metabolites. A total COD concentration of ethanol and acetate accounted for above 73.3% of total soluble microbial products. Linear regression showed that HPR and ethanol production rate were proportionately correlated at all HRTs which could be expressed as y = 0.9821x − 3.5151 (r² = 0.9498). It is meaningful that the proposed recovery of both hydrogen and ethanol from fermentation process can improve energy production rate and economic profit. Results demonstrated that the best energy production rate was 15.50 kJ L⁻¹ h⁻¹, occurred at HRT = 5 h.     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Biohydrogen production from palm oil mill effluent (POME) by two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of carbon sources in POME

The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H₂/mol-sugar and 8.54 mmol H₂/L·h respectively, while for the mesophilic stage were 1.19 mol H₂/mol-sugar and 1.47 mmolH₂/L·h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H₂/L·h to 10.34 mmol H₂/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 °C) and mesophilic (37.0 °C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique.     

Feasibility of biohydrogen production from Gelidium amansii

The feasibility of hydrogen production from red algae was investigated. Galactose, the main sugar monomer of red algae, was readily converted to hydrogen by dark fermentation. The maximum hydrogen production rate and yield of galactose were 2.46 L H₂/g VSS/d and 2.03 mol H₂/mol galactose_added, respectively, which were higher than those for glucose (0.914 L H₂/g VSS/d and 1.48 mol H₂/mol galactose_added). The distribution of soluble byproducts showed that H₂ production was the main pathway of galactose uptake. 5-HMF, the main byproduct of acid hydrolysis of red algae causes noncompetitive inhibition of H₂ fermentation. 1.37 g/L of 5-HMF decreased hydrogen production rate by 50% compared to the control. When red algae was hydrolyzed at 150 °C for 15 min and detoxified by activated carbon, 53.5 mL of H₂ was produced from 1 g of dry algae with a hydrogen production rate of 0.518 L H₂/g VSS/d. Red algae, cultivable on vast tracts of sea by sunlight without any nitrogen-based fertilizer, could be a suitable substrate for biohydrogen production.     

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

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

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

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.     

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.     

Bio-hydrogen production from ground wheat starch by continuous combined fermentation using annular-hybrid bioreactor

Continuous combined fermentation of ground wheat starch was realized in an annular-hybrid bioreactor (AHB) for hydrogen gas production. A mixture of pure cultures of Clostridium beijerinkii (DSMZ-791) and Rhodobacter sphaeroides-RV were used as seed cultures in combined fermentation. The feed contained 5 g L⁻¹ ground wheat with some nutrient supplementation. Effects of hydraulic residence time (HRT) on the rate and yield of hydrogen gas formation were investigated. Steady-state daily hydrogen production decreased but, hydrogen yield increased with increasing HRT. The highest hydrogen yield was 90 ml g⁻¹ starch at HRT of 6 days. Hydrolysis of starch and fermentation of glucose to volatile fatty acids (VFA) were readily realized at all HRTs. However, slow conversion of VFAs to H₂ and CO₂ by photo-fermentation caused accumulation of VFAs in the medium. Specific and volumetric rates of hydrogen formation also decreased with increasing HRT. High hydrogen yields obtained at high HRTs are due to partial fermentation of VFAs by Rhodobacter sp. The system should be operated at HRTs longer than 5 days for effective hydrogen gas formation by the dark and photo-fermentation bacteria.     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

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.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.