Response of H2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

The effects of external factors on both H₂ production and bidirectional Hox-hydrogenase activity were examined in the non-N₂-fixing cyanobacterium Synechocystis PCC 6803. Exogenous glucose and increased osmolality both enhanced H₂ production with optimal production observed at 0.4% and 20 mosmol kg⁻¹, respectively. Anaerobic condition for 24 h induced significant higher H₂ase activity with cells in BG11₀ showing highest activities. Increasing the pH resulted in an increased Hox-hydrogenase activity with an optimum at pH 7.5. The Hox-hydrogenase activity gradually increased with increasing temperature from 30 ^○C to 60 ^○C with the highest activity observed at 70 ^○C. A low concentration at 100 μM of either DTT or β-mercaptoethanol resulted in a minor stimulation of H₂ production. β-Mercaptoethanol added to nitrogen- and sulfur-deprived cells stimulated H₂ production significantly. The highest Hox-hydrogenase activity was observed in cells in BG11₀-S-deprived condition and 750 μM β-mercaptoethanol measured at a temperature of 70 °C; 14.32 μmol H₂ mg chl a⁻¹ min⁻¹.     

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.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

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.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

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.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Hydrogen production from acid hydrolyzed molasses by the hydrogen overproducing Escherichia coli strain HD701 and subsequent use of the waste bacterial biomass for biosorption of Cd(II) and Zn(II)

This study was devoted to investigate production of hydrogen gas from acid hydrolyzed molasses by Escherichia coli HD701 and to explore the possible use of the waste bacterial biomass in biosorption technology. In variable substrate concentration experiments (1, 2.5, 5, 10 and 15 g L⁻¹), the highest cumulative hydrogen gas (570 ml H₂ L⁻¹) and formation rate (19 ml H₂ h⁻¹ L⁻¹) were obtained from 10 g L⁻¹ reducing sugars. However, the highest yield (132 ml H₂ g⁻¹ reducing sugars) was obtained at a moderate hydrogen formation rate (11 ml H₂ h⁻¹ L⁻¹) from 2.5 g L⁻¹ reducing sugars. Subsequent to H₂ production, the waste E. coli biomass was collected and its biosorption efficiency for Cd²⁺ and Zn²⁺ was investigated. The biosorption kinetics of both heavy metals fitted well with the pseudo second-order kinetic model. Based on the Langmuir biosorption isotherm, the maximum biosorption capacities (q_max) of E. coli waste biomass for Cd²⁺ and Zn²⁺ were 162.1 and 137.9 (mg/g), respectively. These q_max values are higher than those of many other previously studied biosorbents and were around three times more than that of aerobically grown E. coli. The FTIR spectra showed an appearance of strong peaks for the amine groups and an increase in the intensity of many other functional groups in the waste biomass of E. coli after hydrogen production in comparison to that of aerobically grown E. coli which explain the higher biosorption capacity for Cd²⁺ or Zn²⁺ by the waste biomass of E. coli after hydrogen production. These results indicate that E. coli waste biomass after hydrogen production can be efficiently used in biosorption technology. Interlinking such biotechnologies is potentially possible in future applications to reduce the cost of the biosorption technology and duplicate the benefits of biological 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.     

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).     

Simultaneous thermophilic hydrogen production and phenol removal from palm oil mill effluent by Thermoanaerobacterium-rich sludge

Thermoanaerobacterium-rich sludge was used for hydrogen production and phenol removal from palm oil mill effluent (POME) in the presence of phenol concentration of 100–1000 mg/L. Thermoanaerobacterium-rich sludge yielded the most hydrogen of 4.2 L H₂/L-POME with 65% phenol removal efficiency at 400 mg/L phenol. Butyric acid and acetic acid were the main metabolites. The effects of oil palm ash, NH₄NO₃ and iron concentration (Fe²⁺) on hydrogen production and phenol removal efficiency from POME by Thermoanaerobacterium-rich sludge was investigated using response surface methodology (RSM). The RSM results indicated that the presence of 0.2 g Fe²⁺/L, 0.3 g/L NH₄NO₃ and 20 g/L oil palm ash in POME could improved phenol removal efficiency, with predicted hydrogen production and phenol removal efficiency of 3.45 L H₂/L-POME and 93%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with hydrogen production and phenol removal efficiency of 3.43 ± 0.12 L H₂/L-POME and 92 ± 1.5%, respectively. Simultaneous hydrogen production and phenol removal efficiency in continuous stirred tank reactor at hydraulic retention time (HRT) of 1 and 2 days were 4.0 L H₂/L-POME with 85% and 4.2 L H₂/L-POME with 92%, respectively. Phenol degrading Thermoanaerobacterium-rich sludge comprised of Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium aciditolerans, Desulfotomaculum sp., Bacillus coagulans and Clostridium uzonii. Phenol degrading Thermoanaerobacterium-rich sludge has great potential to harvest hydrogen from phenol-containing wastewater.     

Co-fermentation of sewage sludge and algae and Fe2+ addition for enhancing hydrogen production

Co-fermentation of sewage sludge and algae was performed for enhancing the hydrogen production, and the effect of Fe²⁺ on co-fermentation process was examined. Results showed that both co-fermentation process and Fe²⁺ addition promoted hydrogen production. Highest hydrogen production of 28 mL/100 mL (14.8 mL H₂/g VS_added) was obtained from the co-fermentation group with 600 mg/L Fe²⁺ addition, which was 2.15 times, 2.00 times and 1.87 times of mono-fermentation of sludge, mono-fermentation of algae, and the co-fermentation group without Fe²⁺ addition. Both volatile solids and protein degradation were stimulated by co-fermentation process. Microbial analysis showed that co-fermentation groups with Fe²⁺ addition enriched Clostridium sensu stricto 13, Clostridium tertium and Terrisporobacter, which were positively correlated with cumulative hydrogen production. This study suggested that the co-fermentation of sludge and algae in the presence of Fe²⁺ could significantly improve the hydrogen production by stimulating the hydrogen-producing metabolism.     

Photo-catalytic hydrogen production over Fe2O3 based catalysts

The hydrogen photo-evolution was successfully achieved in aqueous (Fe_1−xCr_x)₂O₃ suspensions (0 ≤ x ≤ 1). The solid solution has been prepared by incipient wetness impregnation and characterized by X-ray diffraction, BET, transport properties and photo-electrochemistry. The oxides crystallize in the corundum structure, they exhibit n-type conductivity with activation energy of ∼0.1 eV and the conduction occurs via adiabatic polaron hops. The characterization of the band edges has been studied by the Mott Schottky plots. The onset potential of the photo-current is ∼0.2 V cathodic with respect to the flat band potential, implying a small existence of surface states within the gap region. The absorption of visible light promotes electrons into (Fe_1−xCr_x)₂O₃-CB with a potential (∼−0.5 V_SCE) sufficient to reduce water into hydrogen. As expected, the quantum yield increases with decreasing the electro affinity through the substitution of iron by the more electropositive chromium which increases the band bending at the interface and favours the charge separation. The generated photo-voltage was sufficient to promote simultaneously H₂O reduction and SO₃²⁻ oxidation in the energetically downhill reaction (H₂O + SO₃²⁻ → H₂ + SO₄²⁻, ΔG = −17.68 kJ mol⁻¹). The best activity occurs over Fe_1.2Cr_0.8O₃ in SO₃²⁻ (0.1 M) solution with H₂ liberation rate of 21.7 μmol g⁻¹ min⁻¹ and a quantum yield 0.06% under polychromatic light. Over time, a pronounced deceleration occurs, due to the competitive reduction of the end product S₂O₆²⁻.     

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.     

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.     

Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation

Hydrogen formation performances of different anaerobic bacteria were investigated in batch dark fermentation of waste wheat powder solution (WPS). Serum bottles containing wheat powder were inoculated with pure cultures of Clostridium acetobutylicum (CAB), Clostridium butyricum (CB), Enterobacter aerogenes (EA), heat-treated anaerobic sludge (ANS) and a mixture of those cultures (MIX). Cumulative hydrogen formation (CHF), hydrogen yield (HY) and specific hydrogen production rate (SHPR) were determined for every culture. The heat-treated anaerobic sludge was found to be the most effective culture with a cumulative hydrogen formation of 560 ml, hydrogen yield of 223 ml H₂ g⁻¹ starch and a specific hydrogen production rate of 32.1 ml H₂ g⁻¹ h⁻¹.     

Optimization of photo-hydrogen production based on cheese whey spent medium

Cheese whey wastewater diluted to 10 g lactose/L was initially subjected to dark-fermentation by Enterobacter aerogenes MTCC 2822, and the VFAs-rich spent medium (acetic acid 1900 mg/L, butyric acid 537 mg/L, and traces of propionic acid) was subjected to photo-fermentation through enrichment by Ni²⁺ (0–8 μmol/L), Fe²⁺ (0–100 μmol/L) or Mg²⁺ (0–15 mmol/L) in batch mode by Rhodopseudomonas BHU 01 strain. The maximum cumulative H₂ production (144 ml) and yield (58 mmol) was obtained at 4 μmol Ni²⁺/L. Likewise, Fe²⁺ (60 μmol/L) resulted in maximum cumulative H₂ production (139 ml) and yield (56 mmol). Nevertheless, 6 mmol of Mg²⁺ did not significantly affect H₂ production (110 ml) or yield (44 mmol); the latter value in close proximity with the control (37 mmol). The concomitant reduction in COD was maximum (15.61%) for 4 μmol Ni²⁺/L, followed by 15.33% for 60 μmol Fe²⁺/L, and the least for 6 mmol Mg²⁺/L (14.5%). The observations suggest the role of Fe²⁺ and Ni²⁺ in regulation of nitrogenase and hydrogenase, while that of Mg²⁺ mainly in the biosynthesis of photopigment bacteriochlorophyll (Bchl).     

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.