Glycolate photoproduction by free and alginate-entrapped cells of Chlamydomonas reinhardtii

Summary Chlamydomonas reinhardtii cells provide an effective system for glycolate photoproduction, operative during 30 h when they are growing under low CO₂, in the presence of 1 mM aminooxyacetate and 50 M acetazolamide. Glycolate excretion by the cells can proceed for about 4 days if every other 12 h a high CO₂ level is restored in the culture in the absence of inhibitors. The immobilized system in alginate beads has about a twofold higher glycolate photoproduction rate (23 mol·mg chlorophyll (Chl)^–1·h^–1) than free-living cells (12 mol · mg Chl^–1 · h^–1). Offprint requests to: C. Vílchez     

Prolongation of H2 photoproduction by immobilized, sulfur-limited Chlamydomonas reinhardtii cultures

Two approaches to prolong the duration of hydrogen production by immobilized, sulfur-limited Chlamydomonas reinhardtii cells are examined. The results demonstrate that continuous H2 photoproduction can occur for at least 90 days under constant flow of TAP medium containing micromolar sulfate concentrations. Furthermore, it is also possible to prolong the duration of H2 production by cycling immobilized cells between minus and plus sulfate conditions.     

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions

A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.     

Nitrite uptake by Chlamydomonas reinhardtii cells immobilized in calcium alginate

When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g^–1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO _inf2 ^sup– ·mg^–1 Chl·h^–1, the photosynthetic and respiratory activities being about 120 µmol O₂ produced·mg^–1 Chl·h^–1, and 40 µmol O₂ consumed ·mg^–1 Chl·h^–1, respectively. The nitrite uptake activity required CO₂ in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.Correspondence to: C. Vílchez     

Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters

The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions.     

Long-term endurance and selection studies in hydrogen and oxygen photoproduction by Chlamydomonas reinhardtii

Two wild-type strains of Chlamydomonas reinhardtii have been subjected to repeated cycles of anaerobiosis, carbon dioxide deprivation, and irradiation as a means of testing the long-term stability of hydrogen and oxygen photoproduction and the effectiveness of these conditions as selection or adaptation pressures for increasing hydrogen and/or oxygen yields. Simultaneous hydrogen and oxygen photoproduction yields were monitored in each culture for 160 h. The cells were then removed from the reaction chamber and used to inoculate fresh growth medium to produce the culture for the next experiment. This cycle was repeated five times. Yields of hydrogen and oxygen improved after three cycles and declined in the fourth and fifth; unlike the second and third cycles, extended periods of aerobic growth were used for the fourth and fifth cycles. The stability of hydrogen and oxygen photoproduction was greater in the fifth cycle than in any of the previous cycles. These subpopulations had hydrogen and oxygen production rates, at 160 h, which were nearly equal to the rates at the beginning of the fifth-cycle experiments. Time profiles of the relative hydrogen yields from each of the five cycles, prepared at 32, 80, and 120 h, show that the relative yield in each varies with the point in time at which the profile was taken. Chlorophyll retention increased with each successive cycle, indicating selection or adaptation for a more durable population of cells with respect to the light-harvesting component of the photosynthetic apparatus.     

Halotolerance studies onChlamydomonas reinhardtii: glycerol excretion by free and immobilized cells

The freshwater green algaChlamydomonas reinhardtii can tolerate a maximum saline concentration of 200 mM NaCl. In response to this osmotic shock, the cells accumulated during the first 24 h 15% of the total glycerol synthesized as osmoregulatory metabolite, to provide the corresponding osmotic balance. After this period all glycerol synthesized was excreted to the medium, 4 g L^-1 at 120 h in optimal conditions, before cell degradation occurred. This excretion was about 2-fold higher in Ca-alginate entrapped cells in the presence of 250 mM NaCl. It was concluded that immobilized cells may be of biotechnological interest for continuous glycerol photoproduction in air-lift bioreactors.     

Ammonium regulation of urate uptake in Chlamydomonas reinhardtii

Urate was taken up at a negligible rate by Chlamydomonas reinhardtii cells grown on ammonium and transferred to media containing urate plus ammonium or urate plus chloral hydrate or cycloheximide. Addition of ammonium to cells actively consuming urate produced a rapid inhibition of urate uptake whereas the intracellular oxidation of urate was unaffected. Methylammonium but not glutamine or glutamate inhibited urate uptake. Addition of l-methionine-dl-sulfoximine to cells actively consuming urate provoked ammonium excretion, which was accompanied by a rapid inhibition of urate uptake. In cells growing on urate and exhibiting noticeable levels of nitrite-reductase activity, nitrite caused a sudden inhibition of urate uptake whereas nitrate required a time to induce nitrate reductase and to exert its inhibitory effect on uptake. The urate-uptake system did not require urate for induction since the urate-uptake capacity appeared in nitrogen-starved cells. From these results it is concluded that, in Chlamydomonas reinhardtii, ammonium inhibits urate uptake and also acts as co-repressor of the uptake system.     

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.     

Ammonium Ions Control Gametic Differentiation and Dedifferentiation in Chlamydomonas reinhardtii

Effects of various nitrogen-containing compounds on gameticdifferentiation and dedifferentiation in Chlamydomonas reinhardtiiwere studied. Vegetative cells grown either non-synchronouslyor synchronously differentiated to gametes within 8 h in NH₄Cl-freemedium. Addition of arginine did not interfere with gameticdifferentiation although arginine was utilized by the gametesfor the progression of the cell cycle. Fully differentiatedgametes dedifferentiated rapidly to vegetative cells upon feedingwith     

Photoproduction of ammonium by Chlamydomonas reinhardtii cells immobilized in barium alginate: a reactor feasibility study

Summary Chlamydomonas reinhardtii cells immobilized in Ba-alginate beads provide a stable and effective system for photoproducing ammonium from nitrite in a culture medium containing l-methionine-d,l-sulphoximine. The process was studied in either air-lift, fluidized or packed-bed reactors, the last one providing the most suitable system with a volumetric activity of 2700 mol NH _inf+ ^sup4 ·1^–1 per hour.     

Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chlamydomonas reinhardtii

Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chlamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and ¹⁸O₂ discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the alternative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type.     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following l-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used l-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by l-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.Abbreviations HPLC high-performance liquid chromatography - TAP Tris-acetate-phosphate This work was supported by a grant of the CAICYT, Spain. The secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.To whom correspondence should be addressed.     

Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation

Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 10⁶ cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 10³ hygromycin-resistant clones of trasformants per 10⁶ recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

Inhibitor studies on non-photochemical plastoquinone reduction and H(2) photoproduction in Chlamydomonas reinhardtii

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O(2) uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H(2) in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H(2) production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H(2) production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.