Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor

The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of downcomer and riser (A_d/A_r) in range of 0.4–3.2 were considered. Maximum biomass concentration of 1.63 g L⁻¹ in 9 d under light intensity of 120 μE m⁻² s⁻¹ was observed at A_d/A_r of 1.6. The mixing time of the reactors was inversely proportional to A_d/A_r. Maximal H₂ production was found to be 1600 mL L⁻¹ upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L⁻¹), autoclave (600 mL L⁻¹) and HCl treatment (1230 mL L⁻¹). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L⁻¹ as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H₂ production.     

Cd1−xZnxS supported on SBA-16 as photocatalysts for water splitting under visible light: Influence of Zn concentration

Cd_1−xZn_xS solid solutions (x = 0.05–0.3) supported on mesoporous silica SBA-16 substrate with 3D cubic structure were investigated for hydrogen production from water splitting under visible light. The influence of Zn concentration (x) in the Cd_1−xZn_xS solid solution and support morphology were investigated. The bare SBA-16 substrate was synthetized by the hydrothermal method whereas the Cd_1−xZn_xS photocatalysts were prepared by coprecipitation of metal sulfides from aqueous solutions of Cd²⁺ and Zn²⁺ using Na₂S as precipitating agent. An attempt has been made to determine the photocatalyst structures using several techniques including elemental analysis, N₂ adsorption–desorption, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), high resolution transmission electron microscopy (HRTEM), UV–Vis diffuse reflectance spectroscopy (UV–Vis DRS) and Raman spectroscopy. Surface characterization of the samples by XPS indicates that Cd_1−xZn_xS nanoparticles are unevenly distributed on both external surface and within the pore network. An increase of the band gap energy with increasing Zn loading up to x = 0.2 in the Cd_1−xZn_xS solid solution was observed. As a consequence, H₂ evolution increases gradually with an increase of the Zn loading in the photocatalysts from 0.05 to 0.2 wt% being the Cd_0.8Zn_0.2S/SBA-16 system the most active among the catalysts studied. The highest activity of this photocatalyst was explained in terms not only of its large band gap energy but also by the enhancement of the interaction between the particles of solid solution and the SBA-16 substrate.     

Cd1−xZnxS solid solutions supported on ordered mesoporous silica (SBA-15): Structural features and photocatalytic activity under visible light

A series of Cd_1−xZn_xS (x = 0.05–0.3) photocatalysts supported on ordered mesoporous silica (SBA-15) were prepared and investigated for hydrogen production from water splitting under visible light. Textural, structural and surface photocatalyst properties are determined by N₂ adsorption isotherms, UV–vis, Raman and XPS and related to the activity results in hydrogen production. Raman and XRD results indicated a mutual interaction between Cd and Zn, forming nanoparticles of Cd_1−xZn_xS solid solutions. All Cd_1−xZn_xS/SBA-15 samples showed relatively high activities for hydrogen evolution. The hydrogen production rate is found to increase gradually when the zinc concentration on photocatalysts increases from 0.05 to 0.2, achieving a maximum for the photocatalyst with zinc concentration equal to 0.2. Variation in photoactivity is discussed in terms of modification in the conduction band and light absorption ability of Cd_1−xZn_xS particles derived from the changes in the Zn concentration in the Cd_1−xZn_xS solid solution.     

Sugarcane vinasse as substrate for fermentative hydrogen production: The effects of temperature and substrate concentration

The present study aimed to evaluate the hydrogen production of a microbial consortium using different concentrations of sugarcane vinasse (2–12 g COD L⁻¹) at 37 °C and 55 °C. In mesophilic tests, the increase in vinasse concentration did not significantly impact the hydrogen yield (HY) (from 1.72 to 2.23 mmol H₂ g⁻¹ COD_influent) but had a positive effect on the hydrogen production potential (P) and hydrogen production rate (R_m). On the other hand, the increase in the substrate concentration caused a drop in HY from 2.31 to 0.44 mmol H₂ g⁻¹ COD_influent in the tests performed at 55 °C with vinasse concentrations from 2 to 12 g COD L⁻¹. The mesophilic community was composed of different species within the Clostridium genus, and the thermophilic community was dominated by organisms affiliated with the Thermoanaerobacter genus. Not all isolates affiliated with the Clostridium genus contributed to a high HY, as the homoacetogenic pathway can occur.     

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H₂). CO-dependent H₂ production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H₂ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein⁻¹) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein⁻¹). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein⁻¹. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H₂ from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H₂ase for CO-dependent H₂ production.     

Novel photocatalysts based on Cd1−xZnxS/Zn(OH)2 for the hydrogen evolution from water solutions of ethanol

Multiphase photocatalysts Pt/Cd_1−xZn_xS/ZnO/Zn(OH)₂, Pt/Cd_1−xZn_xS/ZnO, and Pt/Cd_1−xZn_xS/Zn(OH)₂ were synthesized by a new two-step technique. The photocatalysts were characterized by a wide range of experimental techniques: X-ray diffraction, high-resolution transmission electron microscopy combined with energy-dispersive X-ray spectroscopy, low-temperature N₂ adsorption/desorption, and UV/VIS spectroscopy. The photocatalytic activity was tested in a batch reactor in the reaction of H₂ evolution from aqueous solutions of ethanol under visible light irradiation (λ > 420 nm). The highest achieved photocatalytic activity was 2256 μmol H₂ per gram of photocatalyst per hour; the highest quantum efficiency was 10.4%. The activity of Pt/Cd_1−xZn_xS/Zn(OH)₂ was higher than that of Pt/Cd_1−xZn_xS/ZnO/Zn(OH)₂ and Pt/Cd_1−xZn_xS/ZnO. The explanation of enhanced activity of zinc–cadmium sulfide/ε-zinc hydroxide based on quantum calculations was suggested.     

Ni (II) and Mg (II) ions as factors enhancing biohydrogen production by Rhodobacter sphaeroides from mineral springs

Various metal ions play a key role in biohydrogen (H₂) production by phototrophic bacteria through incorporation into or stimulating the responsible enzymes and/or related pathways. The Ni (II) and Mg (II) ions effects on growth and H₂ production by Rhodobacter sphaeroides strain MDC6521 isolated from mineral springs in Armenia were established. The highest growth specific rate was obtained with 4–6 μM Ni²⁺ and 5 mM Mg²⁺. pH of the growth medium changed from 7.0 to 9.2–9.4 during the bacterial growth up to 72 h in spite of Ni²⁺ added but pH increased in different manner with Mg²⁺. In the presence of 2–4 μM Ni²⁺ external oxidation-reduction potential (ORP) decreased to more negative values (−800 ± 15 mV). This decrease of ORP indicated ∼2.7-fold enhanced H₂ yield (9.80 mmol L⁻¹) with Ni²⁺ compared with the control (without Ni²⁺). The H₂ yield determined in the medium with Mg²⁺ was ∼2.2 fold higher than that with 1 mM Mg²⁺. These results reveal new regulatory ways to improve H₂ production by R. sphaeroides those were depending on Ni²⁺ and Mg²⁺ of different concentrations.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

The effect of Ni,Fe and Mg concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53

In the present study, the effect of Ni²⁺ (0–10 μmol/l), Fe²⁺ (0–200 μmol/l) and Mg²⁺ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni²⁺ was able to enhance the hydrogen production rate and the hydrogen yield; Fe²⁺ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni²⁺ and Fe²⁺ at a higher concentration inhibited cell growth. When Ni²⁺ and Fe²⁺ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H₂/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg²⁺ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H₂/mol acetate, but it was favorable to cell growth.     

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H₂ is rapidly consumed by the uptake hydrogenase.This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL⁻ (deficient in the uptake hydrogenase), hoxH⁻ (deficient in the bidirectional hydrogenase) and hupL⁻/hoxH⁻ (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N₂ and/or CO₂ addition), light intensity (54 and 152 ??Em⁻²s⁻¹), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium.In every assay, the hupL⁻ and hupL⁻/hoxH⁻ mutants stood out over wild-type cells and the hoxH⁻ mutant. Nevertheless, the hupL⁻ mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 ??Em⁻²s⁻¹ in the light period, with 1 ??M of NiCl₂ supplementation in the culture medium, and under a propane atmosphere.In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO₂ addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO₂ plus 1% of N₂ did not affect positively hydrogen production rate.     

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.     

Optimization of thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

Efficient and stable photocatalytic hydrogen production from water splitting over ZnxCd1–xS solid solutions under visible light irradiation

A simple co-precipitation method was employed to synthesize a series of cubic zinc-blende phase of Zn_xCd_1−xS photocatalysts using Na₂S as the S source. Structural, morphological and optical properties of the samples have been investigated by XRD, SEM, EDS, XRF, ICP, N₂ physisorption and UV–vis diffuse reflectance techniques. The Zn_xCd_1−xS solid solution is not a simple compound mixture of ZnS and CdS, its XRD patterns show new structural peaks instead of mixture of original peaks. The lattice parameter a measured from the XRD patterns of the Zn_xCd_1−xS samples exhibits a slightly nonlinear relationship with the Zn mole fraction, which is slightly inconsistent with Vegard's law, thus suggesting that a nonhomogeneous alloy structure exists in Zn_xCd_1−xS solid solution. The photocatalytic H₂ evolution from water splitting in the sacrificial reagents of 0.25 M Na₂S/0.35 M K₂SO₃ under visible light at 30 °C and 55 °C were also examined in the study. It is found that Zn_xCd_1−xS solid solution with composition x = 0.4–0.5 shows the highest photocatalytic H₂ production performance. The studied Zn_xCd_1−xS exhibits at least 50 h stable photocatalytic activity under outdoor sunlight irradiation.     

Enhanced hydrogen gas production from mixture of beer spent grains (BSG) and distiller's grains (DG) with glycerol by Escherichia coli

Escherichia coli perform mixed acid fermentation and produce hydrogen gas (H₂) as one of the fermentation end products. E. coli can ferment sugars like glucose, xylose and alcohols like glycerol. It has been shown that E. coli has the ability to utilize pretreated organic waste (BSG or DG) or mixtures of it with glycerol and H₂ can be produced. H₂ evolution was maximum when the concentration of BSG was 4% and DG - 10% yielding 1.4 mmol L⁻¹ H₂. H₂ evolution was prolonged to ~24–120 h when mixtures of glycerol and DG or BSG wastes were applied. Moreover, in hycE (lacking large subunit of Hyd-3) or hyfG (lacking large subunit of Hyd-4) single mutants H₂ production was absent compared to wild type suggesting that Hyd-3 and Hyd-4 are responsible for H₂ generation. In addition, multiple mutant enhanced cumulative H₂ production ~3–4 fold. Taken together it can be proposed that BSG or DG wastes either together or in mixture with glycerol can be applied to obtain E. coli biomass and produce bio-H₂. The novel data can be used to further control effectively the application of organic waste resources as a feedstock for developing bio-H₂ production technology.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

The buffer composition impacts the hydrogen production and the microbial community composition in non-axenic cultures

Hydrogen obtained from biomass via dark fermentation is considered a sustainable and clean energy carrier. Batch fermentations with cheese whey powder were performed to assess total hydrogen production (H_max), volumetric hydrogen production rate (VHPR), maximum lactose consumption (S_max), maximum lactose consumption rate (R_max,S), hydrogen molar yield (HMY) and the bacterial species present using two mineral media formulation (A, B). The highest VHPR was 304.8 cm³ dm⁻³ h⁻¹ and the HMY was 1.8 mol mol⁻¹. Medium B yielded around twice the VHPR than the attained with medium A, but HMY only had a slight increment with the use of medium B. The values reached for S_max (17.3 g dm⁻³), H_max (4.863 dm³) and R_max,S (2.7 g dm⁻³ h⁻¹) were also enhanced with medium B. Results suggest that butyrate levels and lower pH are the reasons for diminished hydrogen production with medium A. The microbial communities were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Only one band was observed in the experiments with medium A, the sequence retrieved from this band presented a closest relative match to the sequence from Citrobacter freundii JCM (100% identity); whereas for medium B, three bands were detected. Sequences from these bands presented high homology to sequences from Clostridium perfringens W11 (95% identity), uncultured Lachnospiraceae bacterium clone MS146A1 E12 (100% identity) and Enterobacter cloacae GH1 (100% identity). From the results obtained it is clear that the formulation of culture media had a strong effect on hydrogen production, kinetics and also on the microbial diversity.