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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

“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.     

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.     

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.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Improvement of hydrogen yield of lba-transgenic Chlamydomonas reinhardtii caused by increasing respiration and impairing photosynthesis

The transgenic alga lba of Chlamydomonas reinhardtii yielded H₂ with 50%–180% higher than the control strain. Further experiments showed that photosynthetic rates and photosynthetic reaction center II's photochemical capacities of the transgenic algae obviously decreased 33.4%–85.9% and 30.0%–51.7%, respectively, compared with those of the control. On the contrary, respiration rates of the transgenic algae significantly increased, with 40.0%–200.0% higher than those of the control. Furthermore, starch contents of the transgenic algae were also improved significantly by 79.1%–592.8% compared with the control. Therefore, the reason of H₂ yield improvement of the transgenic alga lba is not only due to its decrease of photosynthetic capacity and increase of the respiration rate, but also due to the metabolic changes related to starch metabolism, photosynthesis and respiration which is possibly caused by hetero-expression of lba gene in chloroplasts of C. reinhardtii, indicating the potential of utilization of lba gene to improve hydrogen yield of micro-green algae.     

Flocculation of wall-deficient cells of Chlamydomonas reinhardtii mutant cw15 by calcium and methanol

Flocculation is a common and inexpensive method for harvesting algae from solution. After nitrogen starvation, it was shown that 83 ± 3% of the wall-deficient cells of the cw 15 mutant of Chlamydomonas reinhardtii flocculated from 12 mL samples within 15 min after the addition of 15 mM calcium chloride at pH 8.4. Only 24 ± 2% of the wildtype strain flocculated under these conditions, thus demonstrating how a simple mutation might facilitate process design. The data suggested that algae grown in waters with similar calcium concentrations (e.g. certain wastewaters) might be harvested through simple pH adjustment. It was also discovered that the addition of small amounts (<5% v/v) of methanol could significantly reduce the calcium needed to achieve flocculation. Within 15 min after addition of 12 mM calcium chloride and 4.6% (v/v) methanol, 83 ± 4% of cw15 cells flocculated. Methanol is fully recoverable by distillation, and its use might enable flocculation without further water salinization when media calcium concentrations fall short of 15 mM. It was further shown that substrates for and/or products of cellular growth affected flocculation adversely. Nearly 81% of cells flocculated from fresh medium compared to only 54% in spent medium.     

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.