Dilution methods to deprive Chlamydomonas reinhardtii cultures of sulfur for subsequent hydrogen photoproduction

Sulfur deprivation of Chlamydomonas reinhardtii cultures gradually inactivates photosynthetic O₂ evolution and leads to the establishment of anaerobiosis in the medium. Sulfur-deprived algal cultures kept under anaerobic conditions will then produce H₂ gas for 3–5 days under continuous illumination. Currently, sulfur deprivation is achieved by mechanical centrifugation of cultures grown in sulfur-replete medium, followed by extensive and costly washing. The cells are finally resuspended in sulfur-free medium. The current study investigates two procedures to deprive algal cultures of sulfur that eliminate the centrifugation step. These procedures involve sulfur deprivation by dilution of sulfur-replete cultures into either sulfur-limited medium or sulfur-free medium. We demonstrate that efficient H₂ photoproduction can be achieved on a timely basis using either procedure. However, the dilution of sulfate-replete algal cultures 1:10 v/v into sulfur-free medium is the most appropriate procedure. These observations serve as the basis for developing an algal H₂-production system that is cheaper, less time-consuming, and less amenable to contamination with other microorganisms than systems employing centrifugation for sulfur deprivation.     

Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii cultures: The effect of the H2 partial pressure

Photoproduction of H₂ gas has been examined in sulfur/phosphorus-deprived Chalmydomonas reinhardtii cultures, placed in photobioreactors (PhBRs) with different gas phase to liquid phase ratios (V_g.p./V_l.p.). The results demonstrate that an increase in the ratio stimulates H₂ photoproduction activity in both algal suspension cultures and in algae entrapped in thin alginate films. In suspension cultures, a 4× increase (from ∼0.5 to ∼2) in V_g.p./V_l.p results in a 2× increase (from 10.8 to 23.1 mmol l⁻¹ or 264–565 ml l⁻¹) in the total yield of H₂ gas. Remarkably, 565 ml of H₂ gas per liter of the suspension culture is the highest yield ever reported for a wild-type strain in a time period of less than 190 h. In immobilized algae, where diffusion of H₂ from the medium to the PhBR gas phase is not affected by mixing, the maximum rate and yield of H₂ photoproduction occur in PhBRs with V_g.p./V_l.p above 7 or in a PhBR with smaller headspace, if the H₂ is effectively removed from the medium by continuous flushing of the headspace with argon. These experiments in combination with studies of the direct inhibitory effect of high H₂ concentrations in the PhBR headspace on H₂ photoproduction activity in algal cultures clearly show that H₂ photoproduction in algae depends significantly on the partial pressure of H₂ (not O₂ as previously thought) in the PhBR gas phase.     

Sustained hydrogen photoproduction by phosphorus-deprived Chlamydomonas reinhardtii cultures

This study demonstrates that, besides sulfur deprivation, sustained H₂ photoproduction in Chlamydomonas reinhardtii cultures can be generated by incubating algae under phosphorus-deprived (−P) conditions. However, phosphorus deficiency in algal cells could not be obtained by resuspension of algae in −P medium, evidently due to a significant reserve of phosphorus in cells. In this study, phosphorus deficiency was accomplished by inoculating the washed algae into the −P medium at low initial cell densities (below 2 mg Chl l⁻¹). After the initial growth period, where cells utilize intracellular phosphorus, algae established anaerobic environment followed by the period of H₂ photoproduction. The maximum H₂ output (∼70 ml l⁻¹) was obtained in cultures with the initial Chl content ∼1 mg l⁻¹. Cultures with Chl above 2 mg l⁻¹ did not produce H₂ gas. The physiological response of algal cultures to phosphorus deprivation demonstrated significant similarities with the response of algae to sulfur depletion.     

Fed-batch operation for bio-H2 production by Rhodopseudomonas palustris (strain 42OL)

Interest in renewable and clean energies such as hydrogen has increased because of the high level of polluting emissions, increasing costs associated with petroleum and the escalating problems of global climate change. In the presence of a light source, a microbial photosynthetic process provides a system for the conversion of some organic compounds into biomass and hydrogen. Using Rhodopseudomonas palustris as a cell-factory, hydrogen photo-evolution was investigated in a photobioreactor (PBR) irradiated either from one or two opposite sides. Irradiating the photobioreactor from only one side, in the presence of malic acid, a reactor hydrogen production of 2.786 l(H₂) PBR⁻¹ was achieved. When the PBR was irradiated from two opposite sides, hydrogen photo-evolution increased to 3.162 l(H₂) PBR⁻¹. Experiments were carried out using inoculum from either the retardation or the exponential growth phases. Using the latter, the highest hydrogen photo-evolution rate based on the bacteriochlorophyll (Bchl) concentration was achieved (3295 μl(H₂) mg (Bchl⁻¹ h⁻¹). The hydrogen to biomass ratio (r_g) was 1.91 l g⁻¹ in the medium containing malic acid and 1.07 l g⁻¹ in that containing acetic acid. It was found that the hydrogen production rate was higher with malic than with acetic acid. Although photobiological hydrogen production cannot furnish alone the greater and greater world requirements of clean renewable energy, it is desirable that photobiological hydrogen technology will grow, in the near future, because photobioreactors for bio-hydrogen production can be positioned in fringe areas without competition with agricultural lands.     

Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii

Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 μM for the hydrogen production. By the re-addition of 30 μM of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H₂ l⁻¹ culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H₂ l⁻¹ culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.     

Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H₂-evolving activities of up to 10 times the O₂-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H₂-metabolism clones following exposure to increasing amounts of O₂, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H₂ following exposure to O₂ concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H₂-production activity once O₂ is removed. These results suggest that O₂-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O₂ when it is actively catalyzing H₂ evolution.     

The photoinduced evolution of O2 and H2 from a WO3 aqueous suspension in the presence of Ce/Ce

This is a report on the production of O₂ and H₂ from photocatalytic and photochemical processes in the WO₃–H₂O–Ce⁴⁺_aq–hν system. The photoproduction of O₂ and H₂ was studied over the range of WO₃ concentrations from 2 to 8 g dm⁻³, and conduction band electron scavenger concentrations 1–20 mM Ce_aq⁴⁺. Medium and high concentrations of the electron scavenger gave mainly O₂ as the main product. Dilute solutions of [Ce_aq⁴⁺]< 2 mM initially produced dioxygen, and then hydrogen after an induction period of 3–4 h. Yields of 140–250 μmol O₂  h⁻¹ and 1–7 μmol H₂ h⁻¹ were obtained and were found to depend on the physical properties and content of WO₃, the concentration of the electron scavenger, illumination period and wavelength, and the radiation geometry. The photoactivity of the suspension was correlated to the level of crystallinity of WO₃ powders. The studied system utilizes WO₃ to accomplish the initial light absorption, charge separation, and production of O₂ and H⁺ from the interaction of water molecules with photogenerated WO₃ valence band holes, in the presence of Ce⁴⁺_aq species as a scavenger of conduction band electrons. This is followed by the evolution of H₂ from a homogeneous photochemical reduction of H⁺ and/or H₂O by photoexcited Ce³⁺_aq, formed from the earlier reduction of Ce⁴⁺_aq. The obtained results show that, with an appropriate design, tungsten trioxide is a promising material that can be used as a photoactive component in energy conversion systems or in environmental photocatalysis, using artificial or solar light.     

Improvement of hydrogen production of Chlamydomonas reinhardtii by co-cultivation with isolated bacteria

Three bacteria, named L2, L3 and L4, were isolated from contaminated cultures of Chlamydomonas reinhardtii strain cc849 in laboratory. The phylogenetic analysis based on 16S rDNA sequences showed that L2, L3 and L4 belonged to genus Stenotrophomonas, Microbacterium and Pseudomonas, respectively. The co-cultivation of isolated L2, L3 and L4 with purified algae, respectively, demonstrated that moderate bacterial concentration did not affect algal growth significantly but improved algal H₂ production obviously. The maximal H₂ yields were gained by the co-culture of algae with L2 or L4, about 4.0 times higher than that of the single algal culture. Increased respiration rate or O₂ consumption was the main reason for the enhancement of H₂ yield of the co-cultures.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

Fermentative hydrogen production with xylose by Clostridium and Klebsiella species in anaerobic batch reactors

Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H₂ and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H₂/mol xylose), 8% (0.5 mol H₂/mol xylose), 10% (0.6 mol H₂/mol xylose) and 14% (0.8 mol H₂/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.     

Feasibility of biohydrogen production from Gelidium amansii

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

Structural stability of chemically delithiated layered (1 − z)Li[Li1/3Mn2/3]O2–zLi[Mn0.5−yNi0.5−yCo2y]O2 solid solution cathodes

Lithium has been chemically extracted from the layered oxide solid solutions Li[Li_1/3Mn_2/3]O₂–(z)Li[Mn_0.5−yNi_0.5−yCo_2y]O₂ (0 ≤ y ≤ 1/2 and 0.25 ≤ z ≤ 0.75) and characterized by X-ray diffraction. The weak super lattice reflections that occur in the parent samples at around 2θ = 20–25° vanish on extracting a significant amount of lithium due to the removal of lithium from the transition metal layer and a consequent loss of the ordering between the Li⁺ and the transition metal ions. Additionally, the chemical delithiation process results in an incorporation of some protons from the chemical delithiation medium into the layered lattice, which has an influence on the structure of the delithiated samples. While the incorporation of a higher concentration (0.4 per formula unit) of protons results in the formation of O1 or P3 phases, delithiated samples with <0.2 protons maintain the initial O3 structure. However, the electrochemically charged samples maintain the initial O3 structure.     

Parameters affecting the growth and hydrogen production of the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has the ability to photosynthetically produce molecular hydrogen (H₂) under anaerobic conditions. It offers a biological route to renewable H₂ production from sunlight and water. Algal growth and H₂ production kinetics must be understood in order to determine appropriate system parameters and develop photobioreactors. Algal biomass should be grown efficiently and economically to attain the high cell densities necessary for H₂ production. The nutrient requirements and process conditions that encourage the growth of dense and healthy algal cultures were explored. Anaerobic conditions were imposed by sulphur deprivation, which requires an exchange of the algal growth medium by centrifugation or dilution. A tubular flow photobioreactor featuring a large surface-to-volume ratio was used to monitor and control the key parameters in the H₂ production process, including pH, dissolved oxygen, optical density, temperature, agitation and light intensity. A cumulative H₂ yield of 3.1 ± 0.3 ml/l of culture was measured.     

Synthesis and characterization of oxygen-rich delafossite CuYO2+x—Application to H2-photo production

Here we report the synthesis and photo electrochemical properties of super oxides CuYO_2.50 and CuYO_2.25 prepared from the delafossite CuYO₂, respectively, by thermal oxidation at 380 °C under O₂-flow and soft chemistry in NaBrO solution (5 N). Their applications as catalysts for H₂ evolution upon visible light were investigated. The oxygen insertion was accompanied by partial oxidation of Cu⁺. For CuYO_2.25, the chemical analyses revealed the presence of mixed valent states containing at least formally an equal number of Cu⁺ and Cu²⁺. The thermal analysis (TGA) under reducing atmosphere indicates that oxygen is inserted in different crystallographic sites, for CuYO_2.25 it exhibits a two-step reduction mechanism with restoration of the parent oxide. In air, CuYO_2+x is thermally stable up to 500 °C above which it undergoes irreversible conversion into Cu₂Y₂O₅. They display p-type behavior ascribed to oxygen insertion and the conduction occurs by hopping mechanism between mixed copper valences. Under illumination, the oxides are stabilized by hole consumption reactions involving SO₃²⁻ and S²⁻ as holes scavengers. The flat-band potentials, lying between 0.17 and 0.26 V_SCE, allow a spontaneous H₂-photo formation. The rate of H₂-evolution is altered by the oxygen insertion and the best photo activity (1.33 μmol h⁻¹ mg⁻¹) was obtained over CuYO_2.25 immersed in S²⁻ solution (0.025 M); CuYO₂ is also reported for a comparison goal. Over time, the photoactivity is slowed down because of the competitive reduction of H₂O with the final products namely S₂O₆²⁻ and S_n²⁻.     

High performance of proton-conducting solid oxide fuel cell with a layered PrBaCo2O5+δ cathode

A dense BaZr_0.1Ce_0.7Y_0.2O_3−δ (BZCY) electrolyte is fabricated on a porous anode by in situ drop-coating method which can lead to extremely thin electrolyte membrane (10 μm in thickness). The layered perovskite structure oxide PrBaCo₂O_5+δ (PBCO) is synthesized by auto ignition process and initially examined as a cathode for proton-conducting IT-SOFCs. The electrical conductivity of PrBaCo₂O_5+δ (PBCO) reaches the general required value for the electrical conductivity of cathode absolutely. The single cell, consisting of PrBaCo₂O_5+δ (PBCO)/BaZr_0.1Ce_0.7Y_0.2O_3−δ (BZCY)/NiO-BaZr_0.1Ce_0.7Y_0.2O_3−δ (BZCY) structure, is assembled and tested from 600 to 700 °C with humidified hydrogen (3% H₂O) as the fuel and air as the oxidant. An open-circuit potential of 1.01 V and a maximum power density of 545 mW cm⁻² at 700 °C are obtained for the single cell, and a low polarization resistance of the electrodes of 0.15 Ω cm² is achieved at 700 °C.     

Performance of Salix viminalis and Populus nigra × Populus maximowiczii in short rotation intensive culture under high irrigation

On a plantation established in 2004 from stem cuttings at a density of 20,000 trees per hectare, we investigated growth and nutritional plant response to a high hydraulic regime for two species (Salix viminalis and Populus nigra × Populus maximowiczii), using a comparative approach with measurements from irrigated and control plots. The plantation was irrigated from June to September 2005 with about 140 mm per day. The equivalent of 120 Kg NO₃–N, 40 Kg P₂O₅–P and 85 Kg K₂O–K per hectare per year was applied by means of irrigation with wastewater. No mortality occurred and stem biomass production of both poplar and willow species were not statistically different on irrigated and control areas. However, S. viminalis revealed to be more tolerant to flooded conditions since these corresponded more closely to its nutritional requirements (foliar concentration of 20 mgN g⁻¹). The capacity of S. viminalis to withstand waterlogged conditions could play an important role in the sustainability of a plantation for the filtration of effluent at low pollutant concentration.     

A new approach for improving microalgal biohydrogen photoproduction based on safe & fast oxygen consumption

Microalgal H₂ photoproduction has the potential of being an affordable method for producing this alternative fuel; however extreme sensitivity of hydrogenase enzyme to photosynthetic O₂ naturally prevents large-scale H₂ production upon illumination. Although a two-phase sulfur deficiency method has been established to deal with this incompatibility, its time and cost demanding, so that this model is not commercially scalable. Despite much research has been conducted, no proper economic alternative for the sulfur deprivation model, with higher or even same productivity and sustainability, has been presented till now. Herein we propose a simple and viable alternative, through introducing a chemical O₂ scavenger system, called oxysorb, to algal cultures.Oxysorb, in non-cytotoxic concentrations (including 50 or 100 mM sodium ascorbate and 5 ppm cupric sulfate) for CC124 as well as pgr5 cultures (containing 30 μg/ml chlorophyll) showed a fast, safe and persisted O₂ removal capacity, initiating H₂ production in the sulfur-containing cultures, either in photoheterotrophic or in autotrophic conditions. Total H₂ production obtained with CC124 and pgr5 cultures, containing 100 mM oxysorb, was 2–5.5 times higher than sulfur-deprived ones (measured in a small closed system). This higher H₂ productivity in the oxysorb approach was achieved due to anoxia establishment with no ROS production and without impacting PSII activity.     

Improvement of photobiological hydrogen production in Chlorococcum minutum using various oxygen scavengers

The depletion of non-renewable energy resources such as fossil fuels urge the human society to concentrate more on renewable energy including production of biological hydrogen (H₂) from algae. Biological hydrogen or biohydrogen is one of the cleanest and efficient alternate energy sources for human needs. It is a well-known fact that hydrogenase (H₂ase) will work in anoxic condition which is the key enzyme in H₂ generation from photosynthetic algae or advanced plants. Keeping in view the significance of anoxic condition, the present study deals with screening of three oxygen scavengers/removers such as sodium sulfite (Na₂SO₃), sodium metabisulfite (Na₂S₂O₅) and sodium dithionite (Na₂S₂O₄) individually along with universal tris-acetate-phosphate (TAP) medium for improvement of biohydrogen production in green alga Chlorococcum minutum (C. minutum) under in vitro conditions. To the best of our knowledge, for the first time improvement in hydrogen production was achieved using sodium sulfite and sodium metabisulfite individually with algal cultures. Efficient photobiological H₂ production was observed at 24 h in C. minutum in the presence of all the three oxygen scavengers when compared to untreated samples via limiting the oxygen levels but output was more with sodium sulfite treatment. Particularly 0.8 mM Na₂SO₃ is best for enhancement of H₂ production at 24 h when compared to other two scavengers and this may be due to high oxidation state and more electron negativity of this compound. Apart from augmentation in H₂ production in this species, present screening may also be helpful for researchers working in the area of biological H₂ generation.     

Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture

Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H₂ from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H₂ yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H₂ mol⁻¹ xylose_consumed and 3.2 ± 0.16 mol-H₂ mol⁻¹ glucose_consumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H₂ yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H₂ mol⁻¹ sucrose_consumed. The effects of stirring speed and different pH levels on growth and H₂ production were studied in the CSABR for highly efficient H₂ production. Growth and H₂ production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H₂ production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H₂ L⁻¹ h⁻¹ at 32, 52, 67, and 86 h, respectively.