Sulfur-deprived Chlamydomonas Research Articles

The hydrogen metabolism of sulfur deprived Chlamydomonas reinhardtii cells involves hydrogen uptake activities

Several species of unicellular microalgae such as the model species Chlamydomonas reinhardtii possess plastid-localized [FeFe]-hydrogenases which, via ferredoxin, can accept electrons from photosynthetic electron transport. Thereby, under specific conditions, these algae light-dependently produce molecular hydrogen (H2), which offers a sustainable way to generate a “green” and efficient fuel. Until today, the most common way to induce sustained H2 production is to deprive Chlamydomonas of macronutrients such as sulfur (S) which results in a downregulation of photosynthetic production of molecular oxygen (O2) and of assimilatory processes. These acclimation responses allow the O2 sensitive algal [FeFe]-hydrogenases to become active and serve as an alternative electron sink of photosynthesis. Despite much progress in the field and a general understanding of the underlying mechanisms, many basic and applied aspects of the photosynthetic H2 metabolism of eukaryotic algae remain to be elucidated. One rarely investigated factor is that microalgae have also been reported to consume H2, especially as a response to high H2 concentrations. Here, we analyzed the H2 uptake activities of S-deprived Chlamydomonas cells incubated in different PBRs providing different gas phase volumes, either in continuous light or in the dark. We show that H2 uptake occurs after prolonged incubation in the light as well as in sudden darkness. Dark-induced H2 uptake can be delayed adding the phosphoribulose kinase inhibitor glycolaldehyde, suggesting a connection to carbohydrate metabolism. The results indicate that PBR setups as well as envisioned outdoor cultivation systems with natural light-dark cycles have to be carefully designed to prevent efficiency losses.

Changes in the Photosystem II complex associated with hydrogen formation in sulfur deprived Chlamydomonas reinhardtii

Redox properties of the acceptor side of Photosystem II were studied during H2 gas production in cells of Chlamydomonas reinhardtii. Flash-induced variable fluorescence changes and thermoluminescence measurements were performed in wild type and Stm6 mutant cells during different stages of sulfur (S)-deprivation. Analysis of the fluorescence decay kinetics indicated that the forward electron transfer on the acceptor side of Photosystem II was dramatically slowed down during the O2 evolution and O2 consumption stages and was completely blocked in the anaerobic stage of S-deprivation, thus, indicating a complete reduction of the PQ-pool. During the H2 formation stage, the forward electron transfer kinetics in the μsec and msec time scale re-appeared indicating partially restored electron flow from QA− to QB and the PQ-pool. Thermoluminescese measurements fully confirmed the fluorescence kinetic analysis. Activation of hydrogenase in the H2 formation stage is responsible for re-oxidation of the PQ pool and reactivation of the electron flow which was found to be faster and more efficient on the Stm6 mutant due to the higher amount of functionally preserved Photosystem II.

Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii1.

Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O2 and H2 production, thus overcoming the O2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae.

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700 (+) in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

The physiological roles and metabolism of ascorbate in chloroplasts

Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well-established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well-known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de-epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen-evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor-side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV-B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes.

Inhibition of photosystem 2 in starch-enriched Chlamydomonas reinhardtii cells prevents the efficient induction of H2 production in sulfur-depleted cultures

In sulfur-deprived Chlamydomonas reinhardtii cells the activity of photosystem 2 (PSII) has been shown to have a crucial role in the photosynthetic production of H2, since it allows the synthesis of internal reserves such as starch. In the present investigation, the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in starch-enriched and sulfur-depleted C. reinhardtii cultures 1) at the very end of the aerobic phase, and 2) soon after the culture started to evolve H2. In the former case, production of H2 on a volumetric basis was completely down-regulated, although starch mobilization was enhanced. In vitro tests showed that the hydrogenase enzyme was active, although its efficiency of utilization in vivo was lowered very soon in the experiment. When an inhibitor of Rubisco such as glycolaldehyde was added under the same conditions, no substantial improvement in H2 production rates was noted. These findings indicate that, aside starch storage, PSII plays an active role in the induction of the H2 production process.

Effects of culture parameters, light, and mixing on H 2 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 O 2 evolution and H 2 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 O 2 evolution and H 2 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 H 2 production.

Interplay among culture parameters, light, mixing, and photobioreactor configuration during H 2 production by sulfur-deprived Chlamydomonas reinhardtii

The interplay among the culture parameters, light intensity and illumination conditions, mechanical mixing conditions, and reactor configurations during O 2 evolution and H 2 production in sulfur-deprived Chlamydomonas reinhardtii cultures is studied in this work. The advective-diffusive reaction equation is used to describe the diffusion and the local biochemical reactions of the species involved in the biological process for H 2 production. Solutions of the diffusion equation are obtained for different boundary conditions. An empirical equation is used to describe the relation between the light attenuation coefficient and the initial chlorophyll concentration, which characterizes both the culture absorption property and the mechanical mixing condition. The dynamics of O 2 evolution and H 2 production in a tubular photobioreactor illuminated under three different light conditions on the boundary is studied for the cultures with different initial chlorophyll concentrations. For the culture with the initial chlorophyll concentration of 18 mg/L, the light attenuation level of K a 0 = 0.125, and the reference light intensity of 236.3 μE m −2 s −1, the results indicate that the maximum H 2 production is 166 ml for the tubular reactor under uniform light illumination, 163 ml for the flat plate reactor under two-side light illumination, 85 and 75 ml for the tubular and the flat plate reactors, respectively, under one-side light illumination.

Dynamics modeling of hydrogen production by sulfur-deprived Chlamydomonas reinhardtii culture in tubular photobioreactor

A heterogeneous hydrogen production system induced by light attenuation across the culture in a photobioreactor and the boundary conditions is studied by solving the advective-diffusive reaction equation (ADRE) used to describe the system. A uniform light intensity is prescribed on the cylindrical surface of the tubular bioreactor and attenuated by Chlamydomonas reinhardtii culture toward the center. The rate constants and the kinetics orders of the S-system based kinetics equations were determined by correlating with the available experimentally measured data. The photobioreactor was operated for 200 h and the dynamics behavior of O 2 evolution and H 2 production were analyzed. The effects of different initial chlorophyll concentrations and quantities of sulfur re-added to the sulfur deprived culture on H 2 production were studied. The results demonstrate that H 2 production decreases with the light attenuation along radial direction. The overall H 2 production increases with the initial cell concentration and the amount of re-added sulfur, respectively, within the simulated range. The modeled results indicate that optimal combination of the culture parameters under the given light intensity and the mixing condition may exist for high H 2 production.

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 H 2 l −1 culture), compared to the control cells (100 ml H 2 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.

“Effect of light intensity and the light: dark cycles on the long term hydrogen production of Chlamydomonas reinhardtii by batch cultures”

Abstract 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 μE 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.

PHENOTYPIC CHARACTERIZATION AND HYDROGEN PRODUCTION IN CHLAMYDOMONAS REINHARDTII QB-BINDING D1-PROTEIN MUTANTS UNDER SULFUR STARVATION: CHANGES IN CHL FLUORESCENCE AND PIGMENT COMPOSITION1

The effects of QB-binding D1-protein mutations on the phenotypic characteristics and on hydrogen production of sulfur-deprived Chlamydomonas reinhardtii P. A. Dang. cultures were investigated. The mutation involved one (D240) or double (D239–40) amino-acid deletions at positions 240 and 239–240, respectively, in the loop connecting helices D and E of the D1 protein. Phenotypic characterization of the mutants showed the following peculiarities as compared to the wildtype (WT): (i) a higher sensitivity to photoinhibition, (ii) a reduced amount of chl per dry weight and per cell, (iii) a higher respiration-to-photosynthesis ratio, (iv) a higher carbohydrate accumulation during the aerobic phase, and (v) a higher synthesis of xanthophyll-cycle pigments. These differences were translated into a 12- to 18-fold higher hydrogen biogas production.

Relationships between H 2 photoproduction and different electron transport pathways in sulfur-deprived Chlamydomonas reinhardtii

In this study the relationships between photosystem (PS) II dependent and independent pathways of H 2 photoproduction, cyclic electron transport around PS I, chloro- and mitorespiration, and transmembrane ΔpH were examined by inhibitor analysis in S deprived Chlamydomonas reinhardtii. The rate of non-photochemical reduction of plastoquinones in photosynthetic membranes was significantly diminished under starvation which may explain the minor contribution of the PS II independent pathway of H 2 photoproduction in starved cells. The suppressive effect of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea on the long-term H 2 photoproduction was shown to be entirely attributed to the inhibition of electron transport in PS II, whereas non-specific interactions did not take place. Ferredoxin-quinone reductase – dependent cyclic electron transport around PS I slowed down H 2 photoproduction more than two fold. This result was related to the competition between ferredoxin-quinone reductase and hydrogenase for the reduced ferredoxin and to the decrease in transmembrane ΔpH induced by the cyclic electron flow. The ΔpH gradient was shown to down regulate the PS II independent pathway of H 2 photoproduction in starved cells.

Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur-deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 micromol photons m(-2) s(-1) in combination with chlorophyll concentrations of 12 and 24 mg L(-1). A high light intensity (140 micromol photons m(-2) s(-1), supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L(-1)) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 micromol photons m(-2) s(-1) (on both sides) and 24 mg L(-1) of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L(-1) in the culture mixed with the stir bar, and rose to about 170 mL L(-1) in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50-100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process.

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

Abstract The photoproduction of H 2 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 H 2 production; (5) a prolonged period of H 2 production. In standard conditions, the mutant produced more than 500 ml of H 2 , 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% H 2 .

S-systems sensitivity analysis of the factors that may influence hydrogen production by sulfur-deprived Chlamydomonas reinhardtii

We built a metabolic map of the hydrogen production process by the microalga Chlamydomonas reinhardtii, mathematically modeled this map in the S-systems formalism, then analyzed the effect of variations in the value of different model parameters on the overall response of the system. The mathematical model exhibited behavior similar to that described in literature for photosynthetic algal hydrogen production by sulfur-deprived algal cultures. This behavior consists of an initial phase during which oxygen is transiently generated and then consumed, followed by an anaerobic phase that is characterized by generation of hydrogen. Our analysis of the effect of independent variables on the hydrogen production process mostly agrees with previous work [Horner J, Wolinsky M. A power-law sensitivity analysis of the hydrogen-producing metabolic pathway in Chlamydomonas reinhardtii. Int J Hydrogen Energy 2002;27: 1251–1255]. Moreover, a more detailed study of the effects of parameter modification (rate constants and kinetic order) indicated that genetic engineering of the hydrogenase expression, activity and stability may lead to increased performance of the process.

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired Photosystem II photochemical activity

Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions

Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production.

06/02771 Demonstration of sustained hydrogen photoproduction by immobilized, sulfur-deprived Chlamydomonas reinhardtii cellsLaurinavichene, T. V. et al. International Journal of Hydrogen Energy, 2006, 31, (5), 659–667

This paper presents a method for the dimensioning of the low-energy District Heating (DH) piping networks operating with a control philosophy of supplying heat in low-temperature such as 55 °C in supply and 25 °C in return regularly while the supply temperature levels are being boosted in cold winter periods. The performance of the existing radiators that were formerly sized with over-dimensions was analyzed, its results being used as input data for the performance evaluation of the piping network of the low-energy DH system operating with the control philosophy in question. The optimization method was performed under different mass flow limitations that were formed with various temperature configurations. The results showed that reduction in the mass flow rate requirement of a district is possible by increasing the supply temperature in cold periods with significant reduction in heat loss from the DH network. Sensitivity analysis was carried out in order to evaluate the area of applicability of the proposed method. Hence varied values of the original capacity and the current capacity of the existing radiators were evaluated with the design temperature values that were defined by two former radiator sizing standards.

Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions

Thus far, all experiments leading to H 2 production by sulfur-deprived cultures of microalga have been done with photoheterotrophic cultures in the presence of acetate, which increases the cost of the H 2 produced. This study demonstrates that sustained H 2 photoproduction by a sulfur-deprived green alga, Chlamydomonas reinhardtii, is possible under strictly photoautotrophic conditions in the absence of acetate or any other organic substrate in the medium. To accomplish this, we used cultures pre-grown with 2% CO 2 under low light conditions ( 25 μ E m - 2 s - 1 ) and also supplemented with CO 2 during S-deprivation along with a special light regime. Maximum H 2 production ( 56.4 ± 16.7 ml l - 1 culture, equal to 56.4 × 10 - 3 m 3 m - 3 culture) was observed with photoautotrophic cultures: (a) supplied with carbon dioxide for the first 24 h of sulfur deprivation, (b) exposed during the O 2-producing stage to high light ( 110 -- 120 μ E m - 2 s - 1 ) , and (c) then exposed to low light ( 20 -- 25 μ E m - 2 s - 1 ) during the O 2-consumption and H 2-production stages.