Hydrogen production by a green alga, Chlamydomonas reinhardtii, in an alternating light/dark cycle

The evolution of H(2) in the dark period of a light/dark cycle by a green alga, Chlamydomonas reinhardtii, was studied with the aim of developing a two/stage biophotolysis system. The algal cells accumulated starch during the growth period in light. When these cells were incubated microaerobically in the dark, hydrogenase activity was induced was induced without an appreciable lag time and therapy H(2) evolution was observed for several hours to more than 10 h, depending upon the amount of added O(2). The cells harvested in the midlogarithmic growth phase were the most efficient in production of H(2) in the dark. H(2) evolution was highly dependent on temperature, but rather incentive to pH values from 5-9. Based on these observations, altering production of O(2) and H(2) was demonstrated repeatedly in a light/dark cycle.     

Xanthophyll cycle induction by anaerobic conditions under low light in Chlamydomonas reinhardtii

The effect of anaerobiosis on the induction of the xanthophyll cycle was investigated in Chlamydomonas reinhardtii. The results showed that, anaerobiosis obtained by either sulfur starvation or by bubbling nitrogen in the culture grown in complete medium induced the xanthophyll cycle even when cultures were exposed to low light conditions. The zeaxanthin content reached 35 mmol mol^?1 Chl a, after 110 h in anaerobic sulfur-starved cultures, and 30 mmol mol^?1 Chl a within 24 h in sulfur replete cultures bubbled with nitrogen. Both starved and non-starved cultures grown under aerobic conditions, did not exhibit any sizeable increase in the zeaxanthin content. Chlorophyll fluorescence measurements revealed a decrease in the maximum photochemical quantum yield of PSII (F_v/F_m) by more than 50 %. The chlorophyll fluorescence kinetics (OJIP) analysis showed a strong rise at the J-step indicating a strong reduction of Q_A. Our findings demonstrated that anaerobiosis in low light exposed cultures induced the xanthophyll cycle through a strong increase of the level of plastoquinone pool reduction, which was associated to the formation of a trans-thylakoid membranes proton gradient, while in dark anaerobic cultures, no appreciable induction of xanthophyll cycle could be observed, despite the sizeable increase in non–photochemical quenching.     

Acclimation of Chlamydomonas reinhardtii to extremely strong light

Photosynthesis Research - Most photosynthetic organisms are sensitive to very high light, although acclimation mechanisms enable them to deal with exposure to strong light up to a point. Here we...     

Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light

As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2, and 0.5) in a biological oxygen monitor (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis–irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 μmol m^?2?s^?1. Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 μmol m^?2?s^?1). At lower duty cycles, maintenance respiration reduced net oxygen yield. At higher duty cycles, photon absorption rate exceeded the maximal photon utilization rate, and, as a result, surplus light energy was dissipated which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity.     

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 mol m^–2 s^–1. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 mol m^–2 s^–1 did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 mol m^–2 s^–1) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 mol m^–2 s^–1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - D1- 32 kDa protein of PS II reaction center - FCCP- carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - KCN- potassium cyanide - PBQ- phenyl-p-benzoquinone - PFD- photon flux density - SHAM- salicylhydroxamic acid NBRI Research Publication No. 431.     

Effect of light/dark cycles on wastewater treatments by microalgae

Chlorella kessleri was cultivated in artificial wastewater using diurnal illumination of 12 h light/12 h dark (L/D) cycles. The inoculum density was 10⁵ cells/mL and the irradiance in light cycle was 45 μmol m² s⁻¹ at the culture surface. As a control culture, another set of flasks was cultivated under continuous illumination. Regardless of the illumination scheme, the total organic carbon (TOC) and chemical oxygen demand (COD) was reduced below 20% of the initial concentration within a day. However, cell concentration under the L/D lighting scheme was lower than that under the continuous illuminating scheme. Thus the specific removal rate of organic carbon under L/D cycles was higher than that under continuous illumination. This result suggested thatC. kessleri grew chemoorganotrophically in the dark periods. After 3 days, nitrate was reduced to 136.5 and 154.1 mg NO₃ ⁻-N/L from 168.1 mg NO₃ ⁻-N/L under continuous illumination and under diurnal cycles, respectively. These results indicate nitrate removal efficiency under continuous light was better than that under diurnal cycles. High-density algal cultures using optimized photobioreactors with diurnal cycles will save energy and improve organic carbon sources removal.     

Metabolite profiling of Chlamydomonas reinhardtii under nutrient deprivation

A metabolite profiling technique for Chlamydomonas reinhardtii cells for multiparallel analysis of low-molecular weight polar compounds was developed. The experimental protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity, and process large sample quantities. As a result of the rapid sampling, extraction, and analysis by gas chromatography coupled to time-of-flight mass spectrometry, more than 800 analytes from a single sample could be measured, of which more than 100 could be identified. Analyte responses could be determined mostly with ses less than 10%. Wild-type cells of C. reinhardtii strain CC-125 subjected to nitrogen-, phosphorus-, sulfur-, or iron-depleted growth conditions develop highly distinctive metabolite profiles. Individual metabolites undergo marked changes in their steady-state levels. Compared to control conditions, sulfur-depleted cells accumulated 4-hydroxyproline more than 50-fold, whereas the amount of 2-ketovaline was reduced to 2% of control levels. The contribution of each compound to the differences observed in the metabolic phenotypes is summarized in a quantitatively rigorous way by principal component analysis, which clearly discriminates the cells from different growth regimes and indicates that phosphorus-depleted conditions induce a deficiency syndrome quite different from the response to nitrogen, sulfur, or iron starvation.     

The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii mutants defective in the chloroplast ATP synthase are highly sensitive to light. The ac46 mutant is affected in the MDH1 gene, required for production or stability of the monocistronic atpH mRNA encoding CF(O)-III. In this and other ATP synthase mutants, we show that short-term exposure to moderate light intensities-a few minutes-induces an inhibition of electron transfer after the primary quinone acceptor of photosystem II (PSII), whereas longer exposure-several hours-leads to a progressive loss of PSII cores. An extensive swelling of thylakoids accompanies the initial inhibition of electron flow. Thylakoids deflate as PSII cores are lost. The slow process of PSII degradation involves the participation of ClpP, a chloroplast-encoded peptidase that is part of a major stromal protease Clp. In the light of the above findings, we discuss the photosensitivity of ATP synthase mutants with respect to the regular photoinhibition process that affects photosynthetic competent strains at much higher light intensities.     

Scale-up aspects of photobioreactors: effects of mixing-induced light/dark cycles

The green micro-algae Chlamydomonas reinhardtiiand Dunaliella tertiolecta were cultivated undermedium-duration square-wave light/dark cycles with acycle time of 15 s. These cycles were used to simulatethe light regime experienced by micro-algae inexternally-illuminated (sunlight) air-lift loopbioreactors with internal draft tube. Biomass yieldin relation to light energy was determined as gprotein per mol of photons (400–700 nm). Between 600and 1200 mol m^-2 s^-1 the yield at a10/5 s light/dark cycle was equal to the yield atcontinuous illumination. Consequently, provided thatthe liquid circulation time is 15 s, a considerabledark zone seems to be allowed in the interior ofair-lift loop photobioreactors (33% v/v) without lossof light utilization efficiency. However, at a 5/10 slight/dark cycle, corresponding to a 67% v/v darkzone, biomass yield decreased. Furthermore, bothalgae, C. reinhardtii and D. tertiolecta,responded similarly to these cycles with respect tobiomass yield. This was interesting because they werereported to exhibit a different photoacclimationstrategy. Finally, it was demonstrated that D.tertiolecta was much more efficient at low (average)photon flux densities (57–370 mol m^-2s^-1) than at high PFDs (> 600 mol m^-2s^-1) and it was shown that D. tertiolectawas cultivated at a sub-optimal temperature (20 ^°C).     

Effects of light on gravitaxis and velocity in Chlamydomonas reinhardtii

The effects of light on gravitaxis and velocity in the bi-flagellated green alga Chlamydomonas reinhardtii were investigated using a real time automatic tracking system. Three distinct light effects on gravitaxis and velocity with parallel kinetics were found. Photosynthetically active continuous red light reversibly enhances the swimming velocity and increases or decreases the precision of gravitaxis, depending on its initial level. Blue light flashes induce fast transient increases in velocity immediately after the photophobic response, and transiently decrease or even reverse negative gravitaxis. The calcium dependence of this response, its fluence-response curve and its spectral characteristics strongly suggest the participation of chlamy-rhodopsin in this effect. The third response, a prolonged activation of velocity and gravitaxis, is also induced by blue light flashes, which can be observed even in calcium-free medium.     

Cr6+胁迫对莱茵衣藻光合作用的影响

以莱茵衣藻(Chlamydomonas reinhardtii)为研究材料,采用氧电极和快速叶绿素a荧光诱导动力学方法研究了不同浓度和时间Cr6+处理对其光合作用的影响.结果表明:当Cr6+浓度大于40 μmol/L时,莱茵衣藻细胞数逐渐下降,而藻细胞变大;表观光合速率成为负值,呼吸作用随Cr6+处理浓度的增加先上升后下降至对照水平;莱茵衣藻有活性放氧复合体比例随Cr6+处理浓度的增加逐渐降低,80 μmol/L Cr6+处理3 d时已下降至13.72%;光合驱动力(DFABS)随Cr6+浓度增加逐步下降,并以DFφPo在DFABS的下降中的贡献最大.研究发现,重金属Cr6+胁迫显著影响莱茵衣藻的光合作用,而对呼吸作用则影响较小;Cr6+主要通过损伤供体侧的放氧复合体以及阻断QA至QB的电子传递而抑制光系统Ⅱ的功能;莱茵衣藻光系统Ⅱ对Cr6+处理比较敏感且存在着多个作用位点,并首先影响反应中心光能捕获效率,其次影响反应中心的活性,最后影响QA-之后的电子传递.     

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.     

Regulation of the Chlamydomonas cell cycle by light and dark

By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.     

A simple model of the growth of phytoplankton populations in light/dark cycles

Phytoplankton populations have been shown to be entrained byalternating periods of light and darkness in natural watersas well as in laboratory cultures. A simple model for the growthof such populations, as reflected by cell division, is presentedhere. The model takes as its structural unit the single cell,using Spudich and Sager's transition point hypothesis for thecoupling between received light and cell cycle progression.A stochastic component is also included to account for cell-to-cellvariability. The model predicts that the characteristics ofcell division patterns in populations entrained by photocyclesdepends mainly on the position of the transition point withinthe cell cycle, rather than on the characteristics of the photocyclicregime. The model simulates successfully the major featuresof observed division patterns of several phytoplankton species.In addition, the model can be used to predict division patternsin high frequency photocycles and during transients inducedby shifts in light regime. Under these conditions, the simulatedpatterns are also consistent with the hypothesis of a circadianclock controlled cell cycle, except in the case of free runningtransients. ¹Present address: Station Biologique Roscoff, CNRS, Roscoff29211, France     

Lipidomic Analysis of Chlamydomonas reinhardtii under Nitrogen and Sulfur Deprivation

Chlamydomonas reinhardtii accumulates lipids under complete nutrient starvation conditions while overall growth in biomass stops. In order to better understand biochemical changes under nutrient deprivation that maintain production of algal biomass, we used a lipidomic assay for analyzing the temporal regulation of the composition of complex lipids in C. reinhardtii in response to nitrogen and sulfur deprivation. Using a chip-based nanoelectrospray direct infusion into an ion trap mass spectrometer, we measured a diversity of lipid species reported for C. reinhardtii, including PG phosphatidylglycerols, PI Phosphatidylinositols, MGDG monogalactosyldiacylglycerols, DGDG digalactosyldiacylglycerols, SQDG sulfoquinovosyldiacylglycerols, DGTS homoserine ether lipids and TAG triacylglycerols. Individual lipid species were annotated by matching mass precursors and MS/MS fragmentations to the in-house LipidBlast mass spectral database and MS2Analyzer. Multivariate statistics showed a clear impact on overall lipidomic phenotypes on both the temporal and the nutrition stress level. Homoserine-lipids were found up-regulated at late growth time points and higher cell density, while triacyclglycerols showed opposite regulation of unsaturated and saturated fatty acyl chains under nutritional deprivation.     

A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of Chlamydomonas reinhardtii (wild type). It was found that quantitative information on the trajectory of motion was required for a meaningful interpretation of the autocorrelation functions. Two models for describing the oscillatory motion of the cell were developed; one based on the instantaneous forward-and-backward motion of the cell, and the other based on a sinusoidal perturbation to the average forward motion. Both models gave satisfactory agreement with the shape of the experimentally measured autocorrelation function, thus making it possible to use this measurement to determine mean progressive swimming velocities in a population of greater than 200 cells.     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).