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.     

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.     

极地雪藻对紫外辐射的生理适应性研究

用不同剂量的紫外线辐射极地雪藻Chlamydomonasnivalis,测定了色素含量、蛋白质和总脂含量以及自由基清除能力的变化。结果表明,极地雪藻具有较强的抵抗紫外辐射的能力,紫外辐射后细胞叶绿素和类胡萝卜素含量增加,增幅随着辐射时间的延长而加大。紫外辐射能使极地雪藻细胞中虾青素含量显著提高。UV-B辐射后,极地雪藻的基本生化成分含量发生了较为明显的变化:蛋白质含量随辐射时间的增长而逐渐降低,辐射8h后降低了18.3%,而总脂含量则逐步增加,辐射8h后增加了32.0%。经过不同时间UV-B辐射培养以后,极地雪藻在有机溶剂系统中的清除自由基能力有所提高。     

衣藻对重金属生物吸附的实验研究

以衣藻为生物吸附材料,研究了它对重金属钴、铜、锌的生物吸附,并用Freundlich等温吸附方程对实验数据进行处理。结果表明衣藻对这三种金属吸附能力大小顺序为锌钴铜;农藻对重金属的吸附能力很强,可用衣藻来处理含重金属的废水。     

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.     

Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii can produce hydrogen gas (H₂) in the presence of the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The addition of 15 μM CCCP to the algal cultures led to 13-fold increase in H₂ photoproduction compared to the control cultures without CCCP treatment. CCCP completely inhibited the photochemical activity of photosystem (PS) II under illumination. In order to better understand metabolic conditions necessary for sustained H₂ production, we have used gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) for metabolomics analysis that is independent of nutritional stress, specifically, sulfur deprivation, which had been used previously to induce H₂ photoproduction. Even 10 min after addition of CCCP, metabolites from many metabolic modules were found drastically decreased, including levels of free amino acids, unsaturated free fatty acids and nucleotides. During prolonged CCCP exposure H₂ production was found to be stable for at least 12 h with a continued increase in levels of free fatty acids. These results indicate that CCCP might become a useful treatment for production of biohydrogen in reactors. The increase in fatty acid production might then be a useful addition for production of carbon-derived biofuels.     

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.     

莱茵衣藻硫胁迫相关microRNA检测及其靶基因分析

采用特异反转录引物,构建莱茵衣藻microRNA(miRNA)的cDNA文库.选用U4核仁小分子RNA(small nucleolar RNA,snoRNA)作为内参,用SYBR(R)green RT-PCR对3种与莱茵衣藻缺硫胁迫反应相关的miRNA进行检测,利用在线平台软件Web MicroRNAs Designer(WMD3)对miRNA靶基因进行预测.结果表明,在缺硫胁迫下,3种miRNA(miRNA1145.2、miRNA1146和miRNA1158)的表达水平均明显上调,与正常培养的莱茵衣藻表达miRNA的相对丰度比值分别为3.11、2.38和3.67.靶基因预测分析表明,miRNA1145.2能影响脂肪酸氧化反应,miRNA1146与辅酶Q代谢相关,miRNA1158可能参与磷酸戊糖途径调控.     

UV-B辐射下NO对衣藻生长及抗氧化酶活性的影响

以衣藻(Chlamydomorm sp.)为实验材料,研究了在紫外线(Uv-B)辐射增强的情况下,外源SNP(硝普钠,N0供体)对其抗氧化酶SOD(超氧化物岐化酶)、CAT(过氧化氢酶)、POD(过氧化物酶)活性的变化以及对衣藻生长曲线的影响.探讨了NO对藻类在辐射休克情况下的生理保护作用.     

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N₂) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N₂ by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.