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.     

Effects of culture parameters, light, and mixing on H2 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 O₂ evolution and H₂ 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 O₂ evolution and H₂ 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 H₂ production.     

Hydrogen production from biological systems under different illumination conditions

Hydrogen is a natural by-product of several microbial driven biochemical reactions, mainly in anaerobic fermentation processes. In addition, certain microorganisms produce enzymes by which H₂ from water may be obtained if an outside energy source, like sunlight, is provided. Biophotolysis is a biological process which involves solar energy and algae clusters to convert water into hydrogen. Algae pigments absorb solar energy and enzymes in the cell act as catalysts to split water into hydrogen and oxygen. There are many research activities studying hydrogen production from biological systems cyanobacteria and green algae and some studies present a complete outline of the main available pathways to improve the photosynthetic H₂ production [1] and [2].Efficiency (energy produced from hydrogen divided by solar energy) of such processes can be estimated up to 10%. This value has to be increased for a large-scale hydrogen production. The effect of different artificial illumination conditions on H₂ production was studied for green algae cultures (Chlamydomonas reinhardtii). Results will be used to design a high-efficiency photobioreactor for a large-scale hydrogen production.     

响应面法优化Chlamydomonas sp.212产胞内多糖发酵工艺及其抑菌活性

通过响应面法优化淡水微藻Chlamydomonas sp.212产胞内多糖的发酵工艺,并采用滤纸片法对其抑菌活性进行研究。经优化后,Chlamydomonas sp.212产胞内多糖发酵工艺的最优参数为Na NO3质量浓度305.01 mg/L、NaCl质量浓度93.66 mg/L、NaHCO_3质量浓度2.12 g/L;在此条件下,其胞内多糖产量为91.182 3 mg/L,比优化前提高了1.6倍。抑菌活性研究结果表明:Chlamydomonas sp.212的胞内多糖对细菌有一定程度的抑制作用,其中对沙门氏菌的抑菌作用最强,对大肠杆菌、白色葡萄球菌、金黄色葡萄球菌的抑制作用较弱;对真菌的抑制作用较弱,仅对黑曲霉有较弱的抑制作用。本研究为筛选新的天然抗菌活性物质及产油微藻的综合开发利用提供了一定的理论依据。     

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.     

SEC2在莱茵衣藻中的表达及免疫学活性分析

通过将金黄色葡萄球菌(Staphylococcus aureus,S.aureus)肠毒素C2(staphylococcal entero-toxin C2,SEC2)基因进行定点突变,获得具有超抗原性的减毒C2基因序列sec2t,把该基因插入含Hsp 70A-RBCS2启动子和RBCS2终止子的pH124载体上,构建莱茵衣藻表达载体pH124sec2t,采用"珠磨法"将该载体转入细胞壁缺陷型莱茵衣藻(Chlamydomonas reinhardtii)CC-849中,经含10μg/mL Zeomy-cin的抗性平板筛选、PCR及RT-PCR分析,获得转基因莱茵衣藻Tran-sec2t.Southern blot和Western blot分析表明,sec2t基因已整合入莱茵衣藻核基因组中,且能表达相对分子质量为26 kDa(1 Da=1 u)的目的蛋白.分别以CC-849和Tran-sec2t两种藻细胞培养液(每毫升106个细胞)喂食Balb/c小鼠,并用流式细胞仪检测分析小鼠CD3+、CD4+和CD8+细胞,结果发现,可表达减毒超抗原SEC2T的转基因莱茵衣藻Tran-sec2t能刺激小鼠T淋巴细胞的产生;与对照相比,CD4+与CD8+细胞数目比值有所降低,但CD3+和CD8+细胞有明显提高.研究结果有助于利用转基因藻生产抗肿瘤制剂和动物免疫增强剂.     

Blue-light induced interaction of LOV domains from Chlamydomonas reinhardtii

The phototropin from Chlamydomonas reinhardtii is a 120 kDa blue light receptor that plays a key role in gametogenesis of this green alga. It comprises two light-sensing domains termed LOV1 and LOV2 (light oxygen and voltage) and a serine/threonine kinase domain. The post-translationally incorporated chromophore is flavin mononucleotide (FMN). Upon absorption of blue light, LOV domains undergo a photocycle that activates a Ser/Thr kinase. The mechanism of this activation is still unknown. We studied the oligomerization of the recombinant LOV1 domain (amino acids 16-133) of C. reinhardtii by means of UV/Vis spectroscopy, size-exclusion chromatography (SEC), and chemical cross-linking with glutardialdehyde. The thermal back-reaction of LOV1 from the signaling state to the dark state as monitored by UV/Vis spectroscopy after an intensive blue light pulse could not be explained by a monoexponential model, although the spectra did not indicate the presence of an additional species. Therefore, we investigated the quaternary structure of the LOV1 domain by size-exclusion chromatography in the dark. This revealed an equilibrium between dimers and higher oligomers (M(W)>200 kDa) under native conditions. No monomers were detected by SEC. However, by analysis of the equilibrium by cross-linking of the protein with glutardialdehyde and subsequent SDS-PAGE, monomers and dimers were identified. Exposure of LOV1 to blue light resulted in a decrease in the monomer/dimer ratio, followed by re-equilibration in the dark. Calculation of the solvent-accessible surface area and the Conolly surfaces of the LOV1 dimers present in the crystal structure support the experimental observation that no mononomers are detected in the native state. A model is presented that accounts for a blue-light-driven change in the quaternary structure of the LOV1 domain and gives hints to the molecular basis of light activation and regulation in LOV-containing proteins.     

荧光探针法测定极地雪藻自由基含量的研究

本文研究利用二氯荧光素双醋酸盐(DCFH-DA)作为荧光探针来测定极地雪藻Chlymydomonas nivalis中自由基的含量.DCFH-DA进入雪藻细胞后由还原型二氯荧光素(DCFH)迅速氧化成氧化型二氯荧光素DCF,其氧化过程的氧化量与自由基的含量成正比,通过荧光分光光度计可直接进行检测.研究表明最佳的测定条件为:荧光探针DCFH-DA用量为60 μmol/L,反应时间1～2.5 h,藻液细胞数为5(106～20(106 个/mL.荧光强度的测定条件为:激发波长:485 nm,发射波长:520 nm.在上述条件下能够稳定、快速地测定藻类细胞中自由基的含量.     

Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O₂ consumption, lower O₂ content and higher H₂ output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O₂ contents and maximal H₂ evolution occurred. The highest H₂ production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H₂ production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H₂ yield by decreasing O₂ content in the medium. This protein had potential to be used in improvement of H₂ production in green algae.