Hydrogenases and photobiological hydrogen production utilizing nitrogenase system in cyanobacteria

We constructed three hydrogenase mutants from Anabaena sp. PCC 7120: ΔhupL (deficient in uptake hydrogenase), ΔhoxH (deficient in bidirectional hydrogenase), and ΔhupL/ΔhoxH (deficient in both genes), and showed that the Δhup and ΔhupL/ΔhoxH produced H₂ at a rate 4–7 times that of wild-type under optimal conditions (Appl. Microbiol. Biotechnol. 58 (2002) 618). We have studied H₂ producing activity of Δhup in more detail. H₂ producing activity of Δhup cells was moderately improved in older cultures when 1% CO₂ was added to the bubbling air. The efficiency of light energy conversion to H₂ by the ΔhupL mutant at its highest H₂ production stage was 1.0–1.6% at an actinic visible light intensity of lower than 50 W/m² under argon atmosphere, and the activity lasted for at least 35 min. At 250 W/m², H₂ producing activity gradually decreased with illumination time.     

Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture

We have analyzed the filamentous cyanobacterium Anabaena PCC 7120 (wildtype) containing one nitrogenase, one uptake hydrogenase and one bidirectional hydrogenase and its hydrogen uptake deficient mutant AMC 414 for their H₂ production capacities. Anabaena PCC 7120 and AMC 414 had similar growth rates in turbidostat mode with increased growth rates at higher light intensity. Rates of C₂H₂ reduction were similar for both strains. In contrast to the wildtype, AMC 414 produced H₂ in a PhotoBioReactor (PhBR) using air as the lifting gas. The rate of H₂ production increased with light intensity and was not even saturated at 456 μEm⁻² s⁻¹. H₂ production increased significantly when replacing the air with argon. The maximal H₂ production during outdoor conditions was recorded using AMC 414 with a peak at 14.9 ml H₂ h⁻¹ l⁻¹. Despite the relatively high production, maximal efficiency of solar energy to H₂ conversion was only 0.042%. A molecular method was developed to analyze the relative abundancies of weight and mutant in competition experiments in the PhBR.     

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.     

Biohydrogen production by Rhodobacter capsulatus Hup mutant in pilot solar tubular photobioreactor

In this study, a pilot solar tubular photobioreactor was successfully implemented for fed batch operation in outdoor conditions for photofermentative hydrogen production with Rhodobacter capsulatus (Hup⁻) mutant. The bacteria had a rapid growth with a specific growth rate of 0.052 h⁻¹ in the batch exponential phase and cell dry weight remained in the range of 1–1.5 g/L throughout the fed batch operation. The feeding strategy was to keep acetic acid concentration in the photobioreactor at the range of 20 mM by adjusting feed acetate concentration. The maximum molar productivity obtained was 0.40 mol H₂/(m³ h) and the yield obtained was 0.35 mol H₂ per mole of acetic acid fed. Evolved gas contained 95–99% hydrogen and the rest was carbon dioxide by volume.     

Extended H2 photoproduction by N2-fixing cyanobacteria immobilized in thin alginate films

Screening of the University of Helsinki Culture Collection for naturally good H₂ producing cyanobacteria recently revealed several promising strains. One of the superior strains is Calothrix 336/3, an N₂-fixing heterocystous filamentous cyanobacterium. Making use of an important feature of the Calothrix 336/3 cells to adhere to the substrate, we applied an immobilization technique to improve H₂ production capacity of this strain. We examined the basic properties of immobilization in Ca²⁺-alginate films in response to the production of H₂ of the Calothrix 336/3 strain and as reference strains we used a model organism Anabaena PCC 7120 and its uptake hydrogenase mutant, ΔhupL, that allow us to compare the responses of different strains to alginate entrapment. Immobilization of the Calothrix 336/3 and ΔhupL mutant cells in Ca²⁺-alginate resulted in prolonged H₂ production over several cycles. Immobilization of the Calothrix 336/3 cells was most successful and production of H₂ could be measured even after 40 days after immobilization.     

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.     

A mixed electronic and protonic conducting hydrogen separation membrane with asymmetric structure

In this work, highly doped ceria with lanthanum, La_0.5Ce_0.5O_2−δ (LDC), are developed as hydrogen separation membrane material. LDC presents a mixed electronic and protonic conductivity in reducing atmosphere and good stability in moist CO₂ environment. LDC separation membranes with asymmetrical structure are fabricated by a cost-saving co-pressing method, using NiO + LDC + corn starch mixture as substrate and LDC as top membrane layer. Hydrogen permeation properties are systemically studied, including the influence of operating temperature, hydrogen partial pressure in feed stream and water vapor in both sides of the membrane on hydrogen permeating fluxes. Hydrogen permeability increases as the increasing of temperature and hydrogen partial pressure in feed gas. Using 20% H₂/N₂ (with 3% of H₂O) as feed gas and dry high purity argon as sweep gas, an acceptable flux of 2.6 × 10⁻⁸ mol cm⁻² s⁻¹ is achieved at 900 °C. The existing of water in both sides of membrane has significant effect on hydrogen permeation and the corresponding reasons are analyzed and discussed.     

Enhancement of hydrogen production by the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005

The efficiency of hydrogen production by different cyanobacterial species depends on several external factors. We report here the factors enhancing hydrogen production by filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Cells adapted to dark-anaerobic conditions produced hydrogen consistent with increased hydrogenase activity when supplemented with Fe²⁺. Stimulation of hydrogen production could be achieved by addition of reductants, either dithiothreitol or β-mercaptoethanol with higher production observed with the latter. Additionally, Fe²⁺ and β-mercaptoethanol added to nitrogen- and sulphur-deprived cells significantly stimulated H₂ production with maximal value of 5.91 ± 0.14 μmol H₂ mg Chla⁻¹ h⁻¹. Glucose and a small increase of osmolality imposed by either NaCl or sorbitol enhanced hydrogen production. High rates of hydrogen production were obtained in cells adapted in nitrogen-deprived medium with neutral and alkaline external pH, significant decrease of hydrogen production occurred under acidic external pH.     

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⁻.     

A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL⁻ strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H₂ evolution under air was observed using an H₂ electrode.     

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).     

A viable antibiotic strategy against microbial contamination in biotechnological production of polyhydroxyalkanoates from surplus whey

The study demonstrates the successful application of vancomycin for inhibiting growth of the contaminant Bacillus cereus in a polyhydroxyalkanoate (PHA) production process with Hydrogenophaga pseudoflava on whey. The minimum inhibiting concentration (MIC) of vancomycin for both strains was calculated in minimal medium H3 as well as in H3 medium supplemented with yeast extract. MIC for B. cereus was 0.57 ??g mL⁻¹ and 1.63 ??g mL⁻¹ in H3 and H3 plus yeast extract respectively. For H. pseudoflava, MIC amounted to 2.8 ??g mL⁻¹ in H3 medium and 6.88 ??g mL⁻¹ in H3 plus yeast extract. The effect of vancomycin on PHA production was minimal or negligible up to a vancomycin concentration of 1 ??g mL⁻¹ in both media. The specific PHA production rates of H. pseudoflava decreased with increasing antibiotics in a minimal medium but in media supplemented with yeast extract the specific PHB production rates increased as the antibiotics concentrations increased.     

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.     

Hydrogen production from fossil and renewable sources using an oxygen transport membrane

Oxygen transport membranes (OTMs) made of mixed ion-electron conductors can be used to increase the production of hydrogen from fossil and renewable sources. This study describes two methods for producing hydrogen with La_0.7Sr_0.3Cu_0.2Fe_0.8O_3−δ (LSCF7328), an OTM material that is easily prepared, exhibits good mechanical properties, and is stable in severe gas conditions. In tests with thin-film (thickness ≈22 μm) LSCF7328 membranes, hydrogen was produced by flowing simulated product streams from CO₂ gasification of coal on one side of the OTM and steam on the other side. In this method, the so-called coal gas on the oxygen-permeate side drives the removal of oxygen from the other side of the OTM, where hydrogen and oxygen are produced by water splitting. With CO (99.5% purity) flowing on the oxygen-permeate side, the hydrogen production rate was measured to be ≈4.7 cm³/min-cm² at 900 °C, indicating that hydrogen can be produced at a significant rate by using product streams from coal gasification. This process also yields a CO₂-rich product stream that is ready for sequestration. In another test, a tubular LSCF7328 was found to increase the hydrogen production from ethanol reforming by supplying high-purity oxygen from air.     

“Effect of light intensity and the light: dark cycles on the long term hydrogen production of Chlamydomonas reinhardtii by batch cultures”

The photomixotrophic hydrogen production was investigated in sulfur deprived Chlamydomonas reinhardtii cultures. The cultures were exposed to continuous illumination of various light intensities in 27-day batches. Light intensity of 70 × 2 ??E m⁻² s⁻¹ was selected for hydrogen production. Subsequent experiments involving 27-day long light:dark cycles were conducted at the selected light intensity. The cycles consisted of hour divisions (h:h; 18:6, 14:10, 12:12) or minute divisions (min:min; 45:15, 35:25, 30:30). The results showed an adverse effect of the light:dark cycles on hydrogen production. All experiments, irrespective of the type of illumination indicated that cultures needed a lag phase for production and the highest hydrogen production was obtained during first 7-10 days of production reaching a peak in the first 5 days.     

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.     

Photocatalytic water splitting into hydrogen and research on synergistic of Bi/Sm with solid solution of Bi–Sm–V photocatalyst

Photocatalyst Bi_1−xSm_xVO₄ were prepared by solid phase reaction and characterized by XRD, UV–visible DRS, BET, and SEM. Bi_1−xSm_xVO₄ showed two structures with the component content. When the composition was above x = 0.3, Bi_1−xSm_xVO₄ were of single phase with tetragonal type and can be regarded as solid solutions of was BiVO₄ and SmVO₄. When Bi_1−xSm_xVO₄ was loaded with 0.3 wt% Pt, the samples showed photocatalytic activities for water decomposition to hydrogen under UV light. Among these catalysts, Bi_0.5Sm_0.5VO₄ showed the best photocatalytic activity for water splitting, which indicated synergistic of Bi/Sm enhanced the photocatalysis efficiency. What's more, Bi_0.5Sm_0.5VO₄ loaded with other co-catalysts was found to act as a photocatalyst for water decomposition to hydrogen and oxygen under UV light irradiation, and the photocatalyst loaded with Pt/Cr₂O₃ had the best photocatalytic property. The amounts of the produced hydrogen and oxygen, respectively, were about 188.25 μmol h⁻¹ g⁻¹ and 95.90 μmol h⁻¹ g⁻¹. This study indicated that the formation of solid solution was the feasible method to adjust energy band and synergistic of Bi/Sm can enhance the photocatalytic activities of water decomposition.