Hydrogen production by Chlamydomonas reinhardtii under light driven sulfur deprived condition

This article explores the possibility of demonstrating sustainable photohydrogen production using Chlamydomonas reinhardtii when grown in sulfur deprived photoautotrophic condition. The hydrogen evolving capability of the algal species was monitored based on alternating light and dark period. Investigation was carried out during the day time in order to exploit the solar energy for meeting the demand of the light period. The results showed that when the reactor was operated at varying photoperiod namely 2, 3 and 4 h of alternating light and dark period, the gas generation was found to be 32 ± 4, 63 ± 7 and 52 ± 5 mL/h, while the corresponding hydrogen content was 47, 86 and 87% respectively. Functional components of hydrogen generation reaction centers were also analyzed, which showed that the PS(I) reaction centers were involved in hydrogen production pathway, as the light absorption by PS(I) was prerequisite for hydrogen generation under sulfur deprived photoautotrophic condition. The findings showed a higher gas yield and hydrogen content under dark period, whereas under light period the gas content was below detectable level for hydrogen due to the reversible hydrogenase reaction.     

The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.     

莱茵衣藻鞭毛内运输蛋白IFT81调控鞭毛组装

利用插入突变的方式获得了一株衣藻不运动突变体ift81,该突变体表现出鞭毛缺失或者短鞭毛的性状.基因序列分析表明,外源基因aphⅧ插入了突变体中IFT81(intraflagellar transport,IFT)基因的第五个外显子内,并导致该外显子原有的52个碱基对被替换.把含有完整IFT81基因的重组质粒导入突变体ift81后,其鞭毛恢复为野生型且可以检测到IFT81-HA融合蛋白的表达,这证明突变体的鞭毛缺陷确实是由于IFT81基因突变所导致.电镜观察显示突变体中鞭毛的显微结构发生改变,免疫荧光实验证实IFT81蛋白主要定位于基体和鞭毛部位.上述结果表明:IFT81蛋白缺失会导致衣藻鞭毛组装缺陷,在鞭毛组装所需蛋白的运输过程中,IFT81蛋白是必不可少的.     

Global Metabolic Regulation of the Snow Alga Chlamydomonas nivalis in Response to Nitrate or Phosphate Deprivation by a Metabolome Profile Analysis

In the present work, Chlamydomonas nivalis, a model species of snow algae, was used to illustrate the metabolic regulation mechanism of microalgae under nutrient deprivation stress. The seed culture was inoculated into the medium without nitrate or phosphate to reveal the cell responses by a metabolome profile analysis using gas chromatography time-of-flight mass spectrometry (GC/TOF-MS). One hundred and seventy-one of the identified metabolites clustered into five groups by the orthogonal partial least squares discriminant analysis (OPLS-DA) model. Among them, thirty of the metabolites in the nitrate-deprived group and thirty-nine of the metabolites in the phosphate-deprived group were selected and identified as “responding biomarkers” by this metabolomic approach. A significant change in the abundance of biomarkers indicated that the enhanced biosynthesis of carbohydrates and fatty acids coupled with the decreased biosynthesis of amino acids, N-compounds and organic acids in all the stress groups. The up- or down-regulation of these biomarkers in the metabolic network provides new insights into the global metabolic regulation and internal relationships within amino acid and fatty acid synthesis, glycolysis, the tricarboxylic acid cycle (TCA) and the Calvin cycle in the snow alga under nitrate or phosphate deprivation stress.     

Pathways of hydrogen photoproduction by immobilized Chlamydomonas reinhardtii cells deprived of sulfur

The green algae Сhlamydomonas reinhardtii entrapped in a thin alginate film have been shown to sustain elevated rates of hydrogen photoproduction under anaerobic incubation in sulfur/phosphorus depleted tris-acetate medium. In the present work we studied mechanisms, underlying hydrogen photoproduction by the immobilized culture, particularly, the roles of PSII and starch accumulation/breakdown. DCMU, a specific inhibitor of electron transport in PSII, is known to suppress hydrogen evolution by circa 80% in suspension cultures of S-deprived C. reinhardtii. In immobilized cells DCMU caused successive stimulatory and inhibitory effects on hydrogen photoproduction, both depending on the deprivation status of the algal cell. The inhibitory effect of DCMU was 25% at 70 h of S deficiency when maximal rates of hydrogen photoproduction were observed. Measurements of the light-induced prompt and delayed chlorophyll fluorescence transients and reflectance at 820 nm (P₇₀₀ redox transitions) revealed very rapid decline of PSII activity in the entrapped S-deprived cells as compared with the suspension culture, whereas PSI suffered less. The immobilized culture showed a high capacity to accumulate starch during early stages of S deprivation and relatively high rates of anaerobic starch degradation during the following hydrogen evolution period. DCMU partly inhibited starch breakdown. Results of the present work brought us to the conclusion that PSII-independent pathway of hydrogen evolution is elevated in the immobilized S-deprived cells rather due to the rapid inactivation of PSII, efficient starch catabolism and non-photochemical PQ reduction.     

Acyl-acyl carrier protein pool dynamics with oil accumulation in nitrogen-deprived Chlamydomonas reinhardtii microalgal cells

Microalgae are potential biofuel feedstocks for production of energy-dense triacylglycerols (TAG). Nitrogen deprivation is known to trigger microalgal TAG accumulation by upregulation of de novo fatty acid (FA) biosynthesis through chloroplast-localized Type II FA synthases (FAS). To gain insights into the associated FAS regulatory mechanisms, we applied a recently reported liquid chromatography–mass spectrometry method to examine acyl-acyl carrier protein (ACP) pool compositional changes of the microalga Chlamydomonas reinhardtii over a nitrogen deprivation time-course. We observed that acyl-ACP pools are highly enriched in acetyl-ACP in nutrient-rich media in photoheterotrophically grown cells. Following shift to nitrogen deprivation, acetyl-ACP markedly decreased, and long-chain palmitoyl (16:0)-, stearoyl (18:0)-, and oleoyl (18:1)-ACPs progressively predominated in acyl-ACP pools in parallel with increases in FA and TAG production. This study shows the utility of microalgal cells to study acyl-ACP pool dynamics to gain insights into plant FA biosynthetic regulation and oil enhancement strategies.