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.     

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.     

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.     

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.     

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.     

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.     

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.     

The effect of landfill leachate on hydrogen production in Chlamydomonas reinhardtii as monitored by PAM Fluorometry

This study investigated the effect of landfill leachate on biomass and biohydrogen production from Chlamydomonas reinhardtii. Maximum biomass and cell viability was recorded in 16% leachate medium with a corresponding growth rate of 927 μg/L chl a d⁻¹ as compared to the control of 688 μg/L chl a d⁻¹. Chlamydomonas cultured in leachate-supplemented medium was subsequently induced to produce 37% more biohydrogen compared to the control culture. The spurge in growth can be a consequence of abundant essential elements in the diluted leachate. Energy Dispersive X-ray analysis of cells in a 16% leachate medium had the highest accumulation of Cr, Mn, Fe, Co, Ni, Mo and Cd. The benefits of the leachate medium were further shown during the hydrogen production phase using Pulse Amplitude Modulated Fluorometry. This period was extended to 8 days in comparison to the control. Leachate therefore increases both the biomass and biohydrogen yield of Chlamydomonas.     

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.     

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.     

NADPH fluorescence as an indicator of hydrogen production in the green algae Chlamydomonas reinhardtii

This study investigated cellular Nicotinamide Adenine Dinucleotide Phosphate (NADPH) fluorescence as a potential indicator of biohydrogen production in Chlamydomonas reinhardtii and a β-NADPH standard. NADPH fluorescence profiles of cultures grown in TAP-S (Tris-acetate phosphate minus sulphur) media, TAP (Tris-acetate phosphate) media and TAP + 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were subsequently compared. Hydrogen production induced from sulphur depletion was found to correlate directly (r = 0.941) with NADPH over the ten day period. The addition of leachate was used to increase hydrogen yields, and subsequently increased the NADPH concentration by 50%–70%. A direct correlation was observed (r = 0.929) between NADPH and hydrogen when the leachate supplemented media was used. As NADPH is the terminal electron acceptor in the photosynthetic chain, results show that NADPH has a pivotal role in hydrogen production as a carrier molecule. Under sulphur depletion, cellular NADPH fluorescence can be used as an indicator of hydrogen production.     

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.     

“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.     

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.     

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin in the stroma. In this work, by analyzing wild type and mutants affected in H₂ production, we show that maximal PSII photosynthetic electron transfer during the first seconds of illumination after a prolonged dark-anaerobiosis period is linearly related to hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging screening protocol allowing to isolate mutants impaired in hydrogenase expression or activity, as well as mutants altered in related metabolic pathways required for energy production in anaerobiosis. Compared to previously described screens for mutants impaired in H₂ production, our screening method is remarkably fast, sensitive and non-invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants were isolated and the most affected one was analyzed and shown to be defective for the hydrogenase HydG assembly factor.     

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.