Demonstration of sustained hydrogen photoproduction by immobilized,sulfur-deprived Chlamydomonas reinhardtii cells

It was demonstrated that immobilized, sulfur-deprived algal cultures can photoproduce H₂${}_2$. After identifying the optimal material and procedures for immobilization of Chlamyodomonas reinhardtii   at high cell density, we examined the effect of liquid mixing, sulfate content, acetate levels and light intensity on the H₂${}_2$-production activity of the culture. Our results indicate that (a) liquid mixing is important to provide homogeneous conditions for the immobilized culture; (b) sulfur deprivation is necessary for hydrogen production by immobilized cultures; and (c) high light intensity decreases H₂${}_2$ production. The maximum total volume of H₂${}_2$ produced by the system (160 ml of reactor volume) was 380 ml over 23 days, and the highest rate of H₂${}_2$ production observed was 45 ml day^-1${}^{-1}$. Cell immobilization significantly increased the duration of the H₂${}_2$-photoproduction phase (up to 4 weeks), maintained specific rates of H₂${}_2$ photoproduction similar to those of suspension cultures and showed potential for large increases in H₂${}_2$ production.     

Hydrogen production by Chlamydomonas reinhardtii under light driven sulfur deprived condition

This article explores the possibility of demonstrating sustainable photohydrogen production using Chlamydomonas reinhardtii when grown in sulfur deprived photoautotrophic condition. The hydrogen evolving capability of the algal species was monitored based on alternating light and dark period. Investigation was carried out during the day time in order to exploit the solar energy for meeting the demand of the light period. The results showed that when the reactor was operated at varying photoperiod namely 2, 3 and 4 h of alternating light and dark period, the gas generation was found to be 32 ± 4, 63 ± 7 and 52 ± 5 mL/h, while the corresponding hydrogen content was 47, 86 and 87% respectively. Functional components of hydrogen generation reaction centers were also analyzed, which showed that the PS(I) reaction centers were involved in hydrogen production pathway, as the light absorption by PS(I) was prerequisite for hydrogen generation under sulfur deprived photoautotrophic condition. The findings showed a higher gas yield and hydrogen content under dark period, whereas under light period the gas content was below detectable level for hydrogen due to the reversible hydrogenase reaction.     

Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures

Unsynchronized Chlamydomonas reinhardtii cells subsequently deprived of sulfur produce H₂ under continuous illumination in the laboratory for 3–4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronize their growth and cell division. While it is clear that only insignificant amounts of H₂ can be produced by sulfur-deprived cells during the night period, little work has been done to examine the effects of the light/dark cycles preceding sulfur deprivation on subsequent H₂ photoproduction. We show that (a) C. reinhardtii cells exhibit synchronized growth and cell division in the presence of acetate, (b) cells with the highest specific rates of H₂ photoproduction also have the highest rates of biomass accumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetate accumulation, but not with H₂ photoproduction. This work shows that it is possible to maximize the production of H₂ by sulfur-depriving synchronized cultures at about 4 h after the beginning of the light period.     

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.     

Enhanced hydrogen production through co-cultivation of Chlamydomonas reinhardtii CC-503 and a facultative autotrophic sulfide-oxidizing bacterium under sulfurated conditions

Photoproduction of H₂ using microalgae has been considered as a promising approach for developing sustainable hydrogen energy. The algae C. reinhardtii CC-503 was co-cultured with a facultative autotroph sulfur-oxidizing bacterium Thuomonas intermedia BCRC 17547 to improve H₂ production. The maximum H₂ production of co-culture at sulfur deficiency conditions was 122 μmol/mg Chl with algae/bacteria ratio as 60:1, which was 2.8-fold higher than that of the pure algal culture. Na₂S₂O₃ treatment can result in a maximum H₂ photoproduction rate of 255 μmol/mg Chl, which was 5.9 and 2.1 times higher than those of pure algae culture and co-culture without Na₂S₂O₃. Co-cultivation under sulphate condition can also significantly increase the biomass, respiratory rate, starch content and hydrogenase activity of C. reinhardtii. By supplement of Na₂S₂O₃, persistent (52 days) H₂ production of bacteria/algae co-culture can be achieved. Our results demonstrated that co-culture of C. reinhardtii CC-503 and bacteria BCRC17547 is a cost-effective strategy for improving photobiological H₂ production.     

Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii can produce hydrogen gas (H₂) in the presence of the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The addition of 15 μM CCCP to the algal cultures led to 13-fold increase in H₂ photoproduction compared to the control cultures without CCCP treatment. CCCP completely inhibited the photochemical activity of photosystem (PS) II under illumination. In order to better understand metabolic conditions necessary for sustained H₂ production, we have used gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) for metabolomics analysis that is independent of nutritional stress, specifically, sulfur deprivation, which had been used previously to induce H₂ photoproduction. Even 10 min after addition of CCCP, metabolites from many metabolic modules were found drastically decreased, including levels of free amino acids, unsaturated free fatty acids and nucleotides. During prolonged CCCP exposure H₂ production was found to be stable for at least 12 h with a continued increase in levels of free fatty acids. These results indicate that CCCP might become a useful treatment for production of biohydrogen in reactors. The increase in fatty acid production might then be a useful addition for production of carbon-derived biofuels.     

Enhanced hydrogen production by means of sulfur-deprived Chlamydomonas reinhardtii cultures grown in pretreated olive mill wastewater

Under sulfur-deprived conditions, the metabolism of Chlamydomonas reinhardtii switches to the photoproduction of hydrogen. This process is sustained by both photosystem II-driven water splitting and by the fermentation of stored carbohydrates. We investigated the possibility of using diluted pretreated olive mill wastewaters (OMW), which contain organic acids and sugars, as a substrate on which to grow Chlamydomonas, in order to obtain suitable biomass to produce hydrogen. The cells grown on a mixture of pretreated OMW and TAP (tris-acetate-phosphate) (50% dilution) were found to be richer in carbohydrates and exhibited a greater production of hydrogen (150 ml H₂ l⁻¹ culture), compared to the control cells (100 ml H₂ l⁻¹ culture). In these cultures, the hydrogen production process was characterized by a shorter aerobic phase and a longer hydrogen-production period. The results offer a useful perspective for the utilization of olive mill wastewaters, which constitute an environmental problem, particularly in Mediterranean areas, and for increasing the output for hydrogen production with Chlamydomonas.     

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.     

Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant

The photoproduction of H₂ was studied in a sulfur-deprived Chlamydomonas reinhardtii D1 mutant that carried a double amino acid substitution. The leucine residue L159 was replaced by isoleucine, and the asparagine N230 was replaced by tyrosine (L159I-N230Y). Phenotypic characterization of the mutant showed some interesting features compared to its wild type, namely: (1) a lower chlorophyll content; (2) a higher photosynthetic capacity and higher relative quantum yield of photosynthesis; (3) a higher respiration rate; (4) a very high conversion of violaxanthin to zeaxanthin during H₂ production; (5) a prolonged period of H₂ production. In standard conditions, the mutant produced more than 500 ml of H₂, that is, more than one order of magnitude greater than its wild type, and about 5-times greater than the CC124 strain that was used for comparison. The better performance of the mutant was mainly the result of a longer production period. Biogas produced contained up to 99.5% H₂.     

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.     

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.     

Hydrogen production by Chlamydomonas reinhardtii in a two-stage process with and without illumination at alkaline pH

This work presents the results of a two-stage (carbon fixation and hydrogen production) experimental study for hydrogen production from microalgae using optical fiber as an internal light source. Effect of absence and presence of light on Chlamydomonas reinhardtii culture’s pH shift is also evaluated. The culture pH value is a function of light intensity; the pH in the alkaline range changes from 7.5 to 9.5 in the presence and absence of optical fiber respectively. The maximum rate of hydrogen production in the presence of exogenic glucose and optical fiber is 6 mL/L_cult/hour, which is higher than other reported values. This study has also revealed that the presence of light reduces the lag time for hydrogen production from 12 to 5 h.     

Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii

Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.     

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.     

Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation

Chlamydomonas reinhardtii UTEX 90 accumulated 1.45 g dry cell weight and 0.77 g starch/L during photosynthetic growth using TAP media at 25 ^°C${}^{°}\mathrm{C}$ in presence of 2% _CO2${\mathrm{CO}}_2$ for 3 days. C. reinhardtii biomass was concentrated and then converted into hydrogen and organic acids by anaerobic fermentation with Clostridium butyricum. Organic acids in the fermentate of algal biomass were consecutively photo-dissimilated to hydrogen by Rhodobacter sphaeroides KD131. In the concentrated algal biomass 52% of the starch was hydrolyzed to 37.1 mmol _H2${\mathrm{H}}_2$/L-concentrated algal biomass and 13.6, 25.5, 7.4 and 493 mM of formate, acetate, propionate, and butyrate, respectively by C. butyricum. R. sphaeroides KD131 evolved 5.72 mmol _H2${\mathrm{H}}_2$ per ml-fermentate of algal biomass under illumination of 8 klux at 30 ^°C${}^{°}\mathrm{C}$. Only 80% of the organic acids, mainly butyrate, were hydrolyzed during photo-incubation. During anaerobic conversion, 2.58 mol _H2/mol${\mathrm{H}}_2/\mathrm{mol}$ starch–glucose was evolved using C. butyricum and then 5.72 mol _H2/L${\mathrm{H}}_2/\mathrm{L}$-anaerobic fermentate was produced by R. sphaeroides KD131. Thus, the two-step conversion process produced 8.30 mol _H2${\mathrm{H}}_2$ from 1 mol starch–glucose equivalent algal biomass via organic acids.     

Hydrogen production with the microalga Chlamydomonas reinhardtii grown in a compact tubular photobioreactor immersed in a scattering light nanoparticle suspension

A new photobioreactor design (110 l) for the biological production of hydrogen with the microalga Chlamydomonas reinhardtii is presented. The photobioreactor (PBR) was made up of 64 tubes (i.d., 27.5 mm, length, 2 m) arranged on an 8 × 8 square pitch cell connected by 64 U-bends for a total length of 133 m. The PBR was contained in a rectangular parallelepiped tank (2.5 × 2 × 2 m) made with isotactic polypropylene, except for the opposite square faces which were made of transparent Plexiglas. The tubes were immersed in a thermostatic water bath and continuously illuminated with artificial light. The culture was circulated with a peristaltic pump. To attain a uniform distribution of light to the cells, we used a suspension of silica nanoparticles that scattered the light supplied by the light bulbs (2 × 2000 W) from the opposite square sides of the photobioreactor. Growth experiments carried out with C. reinhardtii CC124 strain, showed a 23% net increase in the final chlorophyll concentration when the nanoparticle suspension was used. Hydrogen production with the C. reinhardtii strain CC124 was investigated with the new photobioreactor design and carried out using a direct inoculum of sulfur-limited cultures having a residual sulfate concentration below 1 mg l⁻¹. The mean hydrogen output was 3121.5 ± 178.9 ml. The reactor fluid dynamic was investigated, and a tri-dimensional light profile inside the PBR is reported.     

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.     

Hydrogen production by hydrothermal oxidation of FeO under acidic conditions

The production of H₂ by oxidation of FeO, taken here as model compound for steel slags, has been investigated both in pure water and under acidic aqueous conditions in the 373–573 K temperature range. Whereas after 65 h, H₂ yield was negligible in pure water at 423 K, the reaction 3 FeO_(s) + H₂O_(l) → Fe₃O_4(s) + H_2(aq) reached near completion at the same temperature within 10 h in a solution containing 0.05 mol/l acetic acid. Increasing acetic acid concentration by one order of magnitude did not yield significantly more H₂. At identical initial pH, acetic acid was found to be more efficient than oxalic acid and hydrochloric acid at enhancing H₂ production. Acidic conditions increased FeO dissolution kinetics and, consequently, improved H₂ yield. The specific efficiency of acetic acid resides in its thermal stability as well as in the potential of ligand-promoted Fe(II) dissolution. We show that the positive kinetics effect of mild acetic acid solutions over H₂ yield evidenced on FeO does not apply directly to steel slags which buffer the pH to high values due to the presence of large amounts of CaO.