Renewable algal photo H2 production without S control using acetate enriched fermenter effluents

Renewable energy production using microorganisms is one of the challenging issues for environmental sustainability. Algal hydrogen (H₂) production has often been achieved by sulfur (S) and chloride ion (Cl^?) deprivation in a growth medium; however, it may not be realistic to control S or Cl^? concentrations in natural sources (e.g., wastewater). In this study, two different green algal species, Chlamydomonas reinhardtii and Chlorella sorokiniana were selected and their photosynthetic activities were compared with different acetate/Cl^? ratios both in batch and continuous modes. At 150 of acetate/Cl^? ratio, the H₂ production rates were 0.25–0.33 μmol L^?1 min^?1 for C. sorokiniana and 0.20–0.38 μmol L^?1 min^?1 for C. reinhardtii, respectively. The hydrogenase (HydA) reactivation and photosystem II (PSII) inhibitor test revealed that biohydrogen production by algae is due to photosynthetic activity. It was found that maintaining acetate/Cl^? ratios greater than 60–100 leads to continuous O₂ depletion and thus renewable H₂ production for both algal species. Molecular dynamics (MD) simulations of hydrogen bonding between Y_z and His₁₉₀ in PSII supported O₂ inhibition using acetate. Using fermenter effluents, C. sorokiniana and C. reinhardtii showed a successful continuous H₂ production of ~80 μmol L^?1 and ~95 μmol L^?1, respectively, for 15 days.     

“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.     

Continuous photosynthetic biohydrogen production from acetate-rich wastewater: Influence of light intensity

Photo-biohydrogen by microalgae is attractive sustainable energy caused by the utilization of solar energy and water. However, due to oxygen (O₂) sensitive hydrogenase (HydA) activity, effective control of O₂ and light intensity is critical for achieving sustainable photosynthetic hydrogen (H₂) production. Here we demonstrate continuous algal H₂ production using acetate-enriched fermenter effluent, achieving the complete O₂ cessation without sulfur depletion. Average H₂ production of 108 ± 4 μmol L^?1 for Chlamydomonas reinhardtii and 88 ± 7 μmol L^?1 for Chlorella sorokiniana at 100 μmol m^?2 s^?1 were observed for 15 days, respectively. The highest light energy to H₂ energy conversion efficiency (LHCE) of 1.61% for C. reinhardtii and 1.06% for C. Sorokiniana was obtained under low light intensity (50 μmol m^?2 s^?1) but the LHCE decreased with the increase of light intensity followed by photoinhibition, which led to a decrease of HydA activity and H₂ production. Low H₂ production was observed at 50 μmol m^?2 s^?1 under the highest LHCE, in which microalgae exhibited photoinhibition biomass growth kinetics to produce chlorophyll a (Chl a) for electron generation. These results demonstrate that light is a feasible strategy for producing electron for H₂ production under anoxygenic photosynthesis.     

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.     

Inhibition of photosystem 2 in starch-enriched Chlamydomonas reinhardtii cells prevents the efficient induction of H2 production in sulfur-depleted cultures

In sulfur-deprived Chlamydomonas reinhardtii cells the activity of photosystem 2 (PSII) has been shown to have a crucial role in the photosynthetic production of H₂, since it allows the synthesis of internal reserves such as starch. In the present investigation, the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in starch-enriched and sulfur-depleted C. reinhardtii cultures 1) at the very end of the aerobic phase, and 2) soon after the culture started to evolve H₂. In the former case, production of H₂ on a volumetric basis was completely down-regulated, although starch mobilization was enhanced. In vitro tests showed that the hydrogenase enzyme was active, although its efficiency of utilization in vivo was lowered very soon in the experiment. When an inhibitor of Rubisco such as glycolaldehyde was added under the same conditions, no substantial improvement in H₂ production rates was noted. These findings indicate that, aside starch storage, PSII plays an active role in the induction of the H₂ production process.     

Pathways of hydrogen photoproduction by immobilized Chlamydomonas reinhardtii cells deprived of sulfur

The green algae Сhlamydomonas reinhardtii entrapped in a thin alginate film have been shown to sustain elevated rates of hydrogen photoproduction under anaerobic incubation in sulfur/phosphorus depleted tris-acetate medium. In the present work we studied mechanisms, underlying hydrogen photoproduction by the immobilized culture, particularly, the roles of PSII and starch accumulation/breakdown. DCMU, a specific inhibitor of electron transport in PSII, is known to suppress hydrogen evolution by circa 80% in suspension cultures of S-deprived C. reinhardtii. In immobilized cells DCMU caused successive stimulatory and inhibitory effects on hydrogen photoproduction, both depending on the deprivation status of the algal cell. The inhibitory effect of DCMU was 25% at 70 h of S deficiency when maximal rates of hydrogen photoproduction were observed. Measurements of the light-induced prompt and delayed chlorophyll fluorescence transients and reflectance at 820 nm (P₇₀₀ redox transitions) revealed very rapid decline of PSII activity in the entrapped S-deprived cells as compared with the suspension culture, whereas PSI suffered less. The immobilized culture showed a high capacity to accumulate starch during early stages of S deprivation and relatively high rates of anaerobic starch degradation during the following hydrogen evolution period. DCMU partly inhibited starch breakdown. Results of the present work brought us to the conclusion that PSII-independent pathway of hydrogen evolution is elevated in the immobilized S-deprived cells rather due to the rapid inactivation of PSII, efficient starch catabolism and non-photochemical PQ reduction.     

Characterization of cell growth and photobiological H2 production of Chlamydomonas reinhardtii in ASSF industry wastewater

Photobiological H₂ production in microalgae is a promising approach for the development of alternative clean and renewable energy. As a unicellular green alga, Chlamydomonas reinhardtii is regarded as an ideal candidate for sustainable photo-H₂ production. However, growth and photo-H₂ producing are still expensive and energy extensive. Wastewater has been suggested as an economical resource for microalgae growth and biofuels production. In this study, we characterized the cell growth and photo-H₂ production of C. reinhardtii CC503 cultured in waste water from pressing process of fermented sweet sorghum stalks during Advanced Solid State Fermentation (ASSF). The maximal cells concentration reached 8.9 × 10⁶ cells/mL in ASSF wastewater medium (AWM) with the fastest growth rate of 0.19 × 10⁶ cells/h, compared to 18.2 × 10⁶ cells/mL and 0.36 × 10⁶ cells/h in TAP medium and to 1.3 × 10⁶ cells/mL and 0.02 × 10⁶ cells/h in BGII medium respectively. The optimized concentration of wastewater for algae cells growth was determined to be 13.3% (7.5 folds dilution), under which, surprisingly the photosynthetic H₂ evolution was increased by more than 700% compared to the cells grown in TAP medium. This system appears to be a good strategy for the development of an economical microalgal photobiological H₂ production scheme. Finally, the possible mechanism for such an H₂ enhancement was identified as the reduction of PSII activity in AWM grown cells.     

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h^?1), a higher butyric acid production of 6.8 g·L^?1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h^?1, with a constant hydrogen production of 507 mL·h^?1, corresponding to a volumetric rate of 6.10 L·L^?1 d^?1, and a yield of 2.39 mol of H₂ per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.     

Photoelectrocatalytic reduction of CO2 to methanol over CuFe2O4@PANI photocathode

The present study was aimed to convert CO₂ into methanol which not only addresses the potential solution for controlling the CO₂ concentration level in the atmosphere but also offers an alternative approach for the production of renewable energy source. In this perspective, a hybrid photocatalyst, PANI@CuFe₂O₄ was synthesized, characterized and used as a photocathode for photoelectrocatalytic (PEC) reduction of CO₂ to methanol in aqueous medium at an applied potential of ?0.4 V vs NHE under visible light irradiation. The combination of PANI with CuFe₂O₄ greatly increased the PEC CO₂ reduction to methanol owing to enhance the CO₂ chemisorption capacity by the photocathode surface and at the same time facilitated the separation of photogenerated electron-hole (e^?/h⁺) pairs. The incident photon to current efficiency (IPCE) and quantum efficiency (QE) for methanol formation in PEC CO₂ reduction could be achieved as 7.1 and 24.0% respectively. The rate of formation of methanol in PEC CO₂ reduction was found as 49.3 μmol g^?1h^?1 with 73% Faradaic efficiency. Compared to photocatalytic reaction, the PEC results demonstrated that the applied potential could effectively separate the photogenerated e^?/h⁺ pairs and therefore, enhanced the PEC CO₂ reduction activity of the hybrid photocatalyst.     

A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain

Photoproduction of H₂ gas was examined in the Chlamydomonas reinhardtii tla1 strain, CC-4169, containing a truncated light-harvesting antenna, along with its parental CC-425 strain. Although enhanced photosynthetic performance of truncated antenna algae has been demonstrated previously (Polle et al. Planta 2003; 217:49-59), improved H₂ photoproduction has yet to be reported. Preliminary experiments showed that sulfur-deprived, suspension cultures of the tla1 mutant could not establish anaerobiosis in a photobioreactor, and thus, could not photoproduce H₂ gas under conditions typical for the sulfur-deprived wild-type cells (Kosourov et al. Biotech Bioeng 2002; 78:731-40). However, they did produce H₂ gas when deprived of sulfur and phosphorus after immobilization within thin (∼300 μm) alginate films. These films were monitored for long-term H₂ photoproduction activity under light intensities ranging from 19 to 350 μE m⁻² s⁻¹ PAR. Both the tla1 mutant and the CC-425 parental strain produced H₂ gas for over 250 h under all light conditions tested. Relative to the parental strain, the CC-4169 mutant had lower maximum specific rates of H₂ production at low and medium light intensities (19 and 184 μE m⁻² s⁻¹), but it exhibited a 4-times higher maximum specific rate at 285 μE m⁻² s⁻¹ and an 8.5-times higher rate at 350 μE m⁻² s⁻¹ when immobilized at approximately the same cell density as the parental strain. As a result, the CC-4169 strain accumulated almost 4-times more H₂ than CC-425 at 285 μE m⁻² s⁻¹ and over 6-times more at 350 μE m⁻² s⁻¹ during 250-h experiments. These results are the first demonstration that truncating light-harvesting antennae in algal cells can increase the efficiency of H₂ photoproduction in mass culture at high light intensity.     

Numerical investigation of turbulent combustion with hybrid enrichment by hydrogen and oxygen

In this study, the NG + H₂/air + O₂ turbulent flame is numerically investigated using the Computational Fluid Dynamics CFD code. The modulation of combustion and radiation is performed respectively by the Eddy Dissipation Model and the Discrete Ordinate Model. The turbulence modeling is carried out by Shear Stress Transport (SST/k-ω) turbulence model. The H₂ amount in the fuel mixture varies under constant volumetric fuel flow between 0 and 60% and the oxidant is composed by 80% air and 20% pure oxygen. The results obtained show the hydrogen addition to Natural Gas improves the mixing between the reactants, reduces their residence time and reduces the length and thickness of the flame. On the other hand, the hydrogen enrichment minimizes the CO₂ and CO production and increases the NO_x level.     

Investigation of the combined effects of acetate and photobioreactor illuminated fraction in the induction of anoxia for hydrogen production by Chlamydomonas reinhardtii

In the context of hydrogen production by microalgae, the growth of Chlamydomonas reinhardtii was characterized under autotrophic and mixotrophic conditions in a fully controlled photobioreactor (PBR). The combined effect of light transfer conditions, as represented by the illuminated fraction γ, with acetate consumption was observed upon establishment of anoxia. Anoxia was reached in batch cultures when γ was close to 1 (almost fully illuminated culture) in mixotrophic conditions while a value of γ ≈ 0.46 in autotrophic conditions was not sufficient. Based on these results, continuous hydrogen production was established in a cylindrical PBR operated in luminostat with constant illumination and in mixotrophic conditions. Maximum hydrogen gas production was equal to 1.4 ± 0.1 ml_H2 l⁻¹ h⁻¹ for photon flux density of 110 μmol m⁻² s⁻¹ and reactor illuminated fraction of γ = 0.5. Carbon mass balance was realized, emphasizing the necessity to work in strictly autotrophic conditions for hydrogen production with no concomitant CO₂ release.     

Stimulatory effect of ascorbate,the alternative electron donor of photosystem II,on the hydrogen production of sulphur-deprived Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700⁺ and hydrogen evolution.     

Extracting high-purity hydrogen via sodium looping-based formic acid dehydrogenation

Formic acid (FA) has been considered as a prospective hydrogen carrier for its potentials to realize hydrogen storage, transportation, and in-situ supply under mild conditions. However, the application of FA dehydrogenation is limited by its unsatisfactory hydrogen concentration and carbon monoxide selectivity. Herein, a sodium looping-based (Na₂CO₃?NaHCO₃) formic acid dehydrogenation (SLFAD) system is proposed for high-purity hydrogen production with ultra-low CO generation via the Na₂CO₃?NaHCO₃ looping. The SLFAD system consists of three parts, which are FA dehydrogenation reactor (FADR), sorption-enhanced carbon oxide removal reactor (CORR), and sodium-based sorbent regeneration reactor (SSRR). Experimental results proved that no sodium formate and sodium oxalate was formed under NaHCO₃ reduction by H₂. A comprehensive assessment of the system was carried out to preliminary verify the feasibility and optimize the operation parameters of the SLFAD system. Results indicated that a maximum hydrogen concentration of 97.905 vol%, a minimum CO concentration of 11.97 ppm, and a high hydrogen production rate of 0.99989 kmol H₂ h^?1 can be obtained under the conditions of atmospheric pressure, FADR temperature at 80 °C, H₂O/HCOOH = 1.2, CORR temperature at 80 °C, and Na₂CO₃/HCOOH = 1.0.     

Escherichia coli (XL1-BLUE) for continuous fermentation of bioH2 and its separation by polyimide membrane

In this work continuous hydrogen production by Escherichia coli (XL1-BLUE) and its purification by membrane gas separation were studied. Firstly, a kinetic investigation was performed on formate supplemented broth in order to determine exponential growth phase (5–7 h) while the most intense hydrogen fermentation takes place. Furthermore, important process design parameters such as saturation constant and maximal growth rate were calculated (K_S = 0.77 g l⁻¹, μ_max = 0.39 h⁻¹). Afterward, based on the kinetic study, continuous hydrogen fermentations using cultures of E. coli (XL1-BLUE) were carried out in a CSTR reactor configuration applying various hydraulic retention times (HRT) related to both exponential and stationary growth period (5 h, 7 h, 9 h). The results indicated that highest hydrogen yield (0.26 mmol H₂/mmol formate added) and productivity (5.1 mmol H₂ l⁻¹ d⁻¹) could be achieved by applying HRT = 7 h that does not allow the living cells to reach stationary phase. In addition to hydrogen production, the concentration of bioH₂ by polyimide membrane under different operational circumstances was investigated using pure and mixed gases, as well. The results of single gas experiments indicated that increasing the temperature has positive effect on separation efficiency. Moreover, the influence of retentate and feed flow ratio (Q_R/Q_F) was studied applying binary H₂/CO₂ gaseous mixture and it was found that polyimide membrane has high potential for H₂ purification since 18% increase in H₂ concentration and 22% decrease in CO₂ content could be attained in the permeate by a one-step separation process.     

Thermodynamic analysis of low-temperature and high-pressure (cryo-compressed) hydrogen storage processes cooled by mixed-refrigerants

Cryo-compressed hydrogen (CcH₂) is a promising hydrogen storage method with merits of high density with low power consumption. Thermodynamic analysis and comparison of several CcH₂ processes are conducted in this paper, under hydrogen storage conditions of 10–100 MPa at 60–100 K. Mixed-refrigerant J-T (MRJT), nitrogen/neon reverse Brayton (RBC) and hydrogen expansion are employed for cooling hydrogen, respectively. Combined CcH₂ processes such as MRJT + neon-RBC are proposed to reach higher CcH₂ density at lower temperatures (<80 K). It was indicated that the specific power consumptions (SPC) of MRJT processes are obviously lower than those of nitrogen/neon-RBC or hydrogen expansion processes. For a typical storage condition of 50 MPa at 80 K, MRJT CcH₂ process could achieve hydrogen density of 71.59 kg m^?3, above liquid hydrogen. While its SPC of 6.42 kWh kg^?1 is about 40% lower than current dual-pressure Claude hydrogen liquefaction processes (10.85 kWh kg^?1).     

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.     

Dark fermentative biohydrogen production using pretreated Scenedesmus obliquus biomass under an integrated paradigm of biorefinery

In recent times, biohydrogen production from microalgal feedstock has garnered considerable research interests to sustainably replace the fossil fuels. The present work adapted an integrated approach of utilizing deoiled Scenedesmus obliquus biomass as feedstock for biohydrogen production and valorization of dark fermentation (DF) effluent via biomethanation. The microalgae was cultivated under different CO₂ concentration. CO₂-air sparging of 5% v/v supported maximum microalgal growth and carbohydrate production with CO₂ fixation ability of 727.7 mg L^?1 d^?1. Thereafter, lipid present in microalgae was extracted for biodiesel production and the deoiled microalgal biomass (DMB) was subjected to different pretreatment techniques to maximize the carbohydrate recovery and biohydrogen yield. Steam heating (121 °C) in coherence with H₂SO₄ (0.5 N) documented highest carbohydrate recovery of 87.5%. DF of acid-thermal pretreated DMB resulted in maximum H₂ yield of 97.6 mL g^?1 VS which was almost 10 times higher as compared to untreated DMB (9.8 mL g^?1 VS). Subsequent utilization of DF effluent in biomethanation process resulted in cumulative methane production of 1060 mL L^?1. The total substrate energy recovered from integrated biofuel production system was 30%. The present study envisages a microalgal biorefinery to produce biohydrogen via DF coupled with concomitant CO₂ sequestration.