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.     

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.     

Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii

The direct relationship between hydrogenase gene conformation and its function in green alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the gene region is present.     

Overexpression of Ferredoxin,PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H₂O₂ levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H₂O₂ and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.     

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.     

Integrated biological hydrogen production

Biological systems offer a variety of ways by which to generate renewable energy. Among them, unicellular green algae have the ability to capture the visible portion of sunlight and store the energy as hydrogen (H₂). They hold promise in generating a renewable fuel from nature's most plentiful resources, sunlight and water. Anoxygenic photosynthetic bacteria have the ability of capturing the near infrared emission of sunlight to produce hydrogen while consuming small organic acids. Dark anaerobic fermentative bacteria consume carbohydrates, thus generating H₂ and small organic acids. Whereas efforts are under way to develop each of these individual systems, little effort has been undertaken to combine and integrate these various processes for increased efficiency and greater yields. This work addresses the development of an integrated biological hydrogen production process based on unicellular green algae, which are driven by the visible portion of the solar spectrum, coupled with purple photosynthetic bacteria, which are driven by the near infrared portion of the spectrum. Specific methods have been tested for the cocultivation and production of H₂ by the two different biological systems. Thus, a two-dimensional integration of photobiological H₂ production has been achieved, resulting in better solar irradiance utilization (visible and infrared) and integration of nutrient utilization for the cost-effective production of substantial amounts of hydrogen gas. Approaches are discussed for the cocultivation and coproduction of hydrogen in green algae and purple photosynthetic bacteria entailing broad utilization of the solar spectrum. The possibility to improve efficiency even further is discussed, with dark anaerobic fermentations of the photosynthetic biomass, enhancing the H₂ production process and providing a recursive link in the system to regenerate some of the original nutrients.     

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.     

Effects of culture parameters, light, and mixing on H2 production by sulfur-deprived Chlamydomonas reinhardtii in flat plate reactor

Based on advective-diffusive reaction equation for inhomogeneous biochemical system and an empirical equation for light attenuation coefficient, the interplay among culture parameters, light intensity and illumination condition, and mechanical mixing condition during O₂ evolution and H₂ production in a flat plate photobioreactor with sulfur-deprived Chlamydomonas reinhardtii culture is modeled in this work. Four initial chlorophyll concentrations, two light attenuation levels, and two illumination conditions were modeled to study their effects on the dynamics of O₂ evolution and H₂ production. The results indicate that two side illumination is the best design for light penetration into a flat plate reactor. While for single side illumination condition, an optimal combination of the initial cell concentration, light intensity, and reactor width may have to be considered for high H₂ production.