Yuanguang Li

Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors

BackgroundMicroalgae can accumulate considerable amounts of lipids under different nutrient-deficient conditions, making them as one of the most promising sustainable sources for biofuel production. These inducible processes provide a powerful experimental basis for fully understanding the mechanisms of physiological acclimation, lipid hyperaccumulation and gene expression in algae. In this study, three nutrient-deficiency strategies, viz nitrogen-, phosphorus- and iron-deficiency were applied to trigger the lipid hyperaccumulation in an oleaginous Chlorella pyrenoidosa. Regular patterns of growth characteristics, lipid accumulation, physiological parameters, as well as the expression patterns of lipid biosynthesis-related genes were fully analyzed and compared.ResultsOur results showed that all the nutrient stress conditions could enhance the lipid content considerably compared with the control. The total lipid and neutral lipid contents exhibit the most marked increment under nitrogen deficiency, achieving 50.32% and 34.29% of dry cell weight at the end of cultivation, respectively. Both photosynthesis indicators and reactive oxygen species parameters reveal that physiological stress turned up when exposed to nutrient depletions. Time-course transcript patterns of lipid biosynthesis-related genes showed that diverse expression dynamics probably contributes to the different lipidic phenotypes under stress conditions. By analyzing the correlation between lipid content and gene expression level, we pinpoint several genes viz. rbsL, me g6562, accA, accD, dgat g2354, dgat g3280 and dgat g7063, which encode corresponding enzymes or subunits of malic enzyme, ACCase and diacylglycerol acyltransferase in the de novo TAG biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification.ConclusionThis study provided us not only a comprehensive picture of adaptive mechanisms from physiological perspective, but also a number of targeted genes that can be used for a systematic metabolic engineering. Besides, our results also represented the feasibility of lipid production through trophic transition cultivation modes, throwing light on a two-stage microalgal lipid production strategy with which heterotrophy stage provides sufficient robust seed and nitrogen-starvation photoautotrophy stage enhances the overall lipid productivity.

Novel flat-plate photobioreactors for microalgae cultivation with special mixers to promote mixing along the light gradient

Novel flat-plate photobioreactors (PBRs) with special mixers (type-a, type-b, and type-c) were designed based on increased mixing degree along the light gradient. The hydrodynamic and light regime characteristic of the novel PBRs were investigated through computational fluid dynamics. Compared with the control reactor without mixer, the novel reactors can effectively increase liquid velocity along the light gradient, the frequency of light/dark (L/D) cycles, and the algal growth rates of Chlorella pyrenoidosa. The maximum biomass concentrations in type-a, type-b, and type-c reactors were 42.9% (1.3 g L(-1)), 31.9% (1.2 g L(-1)), and 20.9% (1.1 g L(-1)) higher than that in the control reactor (0.91 g L(-1)), respectively, at an aeration rate of 1.0 vvm. Correlation analysis of algal growth rate with the characteristics of mixing and light regime shows the key factors affecting algal photoautotrophic growth are liquid velocity along the light gradient and L/D cycles rather than the macro-mixing degree.

Manipulating the pyruvate dehydrogenase bypass of a multi‐vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production

Aims:  To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi‐vitamin auxotrophic yeast Torulopsis glabrata.

Enhanced lipid productivity of Chlorella pyrenoidosa through the culture strategy of semi-continuous cultivation with nitrogen limitation and pH control by CO2.

Microalgae cultivation with high lipid productivity has received much attention in recent years owing to the economic potential of biofuels production and CO2 emission reduction. Previous studies had reported that the ways of pH-regulation, nutrition-limitation and semi-continuous culture mode can either increase the cells growth rate or promote lipid accumulation. In this study, the novel culture strategy of integrating both nutrition limitation and pH-regulation by CO2 in a semi-continuous cultivation was investigated for enhancing the lipid productivity. In the batch culture, the best growth performance was achieved by controlling pH at 7; lipid contents of the cells can be increased under the nitrogen-limitation conditions. The maximum lipid productivity of 115 mg L(-1) d(-1) was achieved in the novel culture strategy of semi-continuous cultivation with nitrogen-limitation and pH-regulation by CO2, which was 3.64-fold higher than that in the batch culture without pH control and nitrogen limitation.

A novel method using CFD to optimize the inner structure parameters of flat photobioreactors

The relationship between growth rates of algae and structure parameters of closed flat-panel photobioreactors was investigated. Ad/Ar (cross-section area of the downcomer/cross-section area of the riser), h0 (clearance from the upper edge of the baffles to the water level), and h1 (clearance from the lower edge of the baffles to the bottom of the reactor) were selected as the inner structure parameters. CFD (Computational Fluid Dynamics) was used to simulate the hydrodynamic parameter (TKE)d (turbulence kinetic energy of the downcomers) and the secondary parameters ε (ratio between td and tc), tc (cycle time), and DZ (dead zones), which were deduced from the hydrodynamic parameters mentioned above. The effects of (TKE)d, ε, tc, DZ, and the inner structure parameters on cell growth of Isochrysis galbana 3011 were analyzed using data collected in 15-L airlift flat-panel photobioreactors. A model was developed to predict algae cell growth based on these inner structure parameters, thereby providing a new method for photobioreactor optimization. Validity of the model was confirmed by experimental data of I. galbana 3011 cultured in 15-L and 300-L photobioreactors, respectively. Finally, the prospect of applying CFD to photobioreactor optimization was discussed.

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

The versatile chlorophyta Chlorella pyrenoidosa provides genomic insights into the trophic diversity and metabolic dynamics. The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

Enhancement of microalgal biomass and lipid productivities by a model of photoautotrophic culture with heterotrophic cells as seed.

For overcoming the long period of seed cultured photoautotrophically and inadequate cell supply for the inoculation of microalgae photoautotrophic cultivation, a model for the photoautotrophic culture of three Chlorella species with heterotrophic cells as seed was investigated. The model can not only take advantages of rapid cell growth in heterotrophic process for preparation of cells as seed but also increase the biomass and lipid productivities of the microalgae cultivated photoautotrophically. The results showed that biomass productivities of Chlorella pyrenoidosa, Chlorella ellipsoidea and Chlorella vulgaris cultured by heterotrophy were 20.9, 26.9 and 25.2 times higher than those by photoautotrophy in seed culturing period. In the subsequent photoautotrophic culture, the biomass and lipid productivities of C. pyrenoidosa, C. ellipsoidea and C. vulgaris with heterotrophic seed were 1.91, 1.51, 1.48 and 1.66, 1.37, 1.42 times higher than those with photoautotrophic seed. Furthermore, the culture model was also carried out successfully outdoor.

Changes of biomass, lipid content and fatty acids composition under a light-dark cyclic culture of Chlorella pyrenoidosa in response to different temperature.

For outdoor culture with light-dark cycle, the biomass and lipid losing at night resulted in lowering the biomass and lipid productivity. Previous studies focused on the contents of carbohydrate and protein in response to temperature for production of animal feed and nutritional supplements. In this study, the effects of temperature on the variations of biomass concentration, lipid content and fatty acids composition for production of biofuels were investigated under a light-dark cyclic culture. The results showed that 30 °C was the optimal daytime temperature for achieving high biomass and lipid; raising daytime temperature can lessen night biomass loss and stimulate lipid accumulation. Subsequently, outdoor culture strategy has been improved: keeping culture broth no less than 30 °C during the daytime. Consequently, the net biomass and lipid productivity were increased by 37.8% and 44.9% when compared to the former culture process in the same outdoor climatic conditions.

Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of Chlorella ellipsoidea

BackgroundThe lipid content of microalgae is regarded as an important indicator for biodiesel. Many attempts have been made to increase the lipid content of microalgae through biochemical and genetic engineering. Significant lipid accumulation in microalgae has been achieved using biochemical engineering, such as nitrogen starvation, but the cell growth was severely limited. However, enrichment of lipid content in microalgae by genetic engineering is anticipated. In this study, GmDof4 from soybean (Glycine max), a transcription factor affecting the lipid content in Arabidopsis, was transferred into Chlorella ellipsoidea. We then investigated the molecular mechanism underlying the enhancement of the lipid content of transformed C. ellipsoidea.ResultsWe constructed a plant expression vector, pGmDof4, and transformed GmDof4 into C. ellipsoidea by electroporation. The resulting expression of GmDof4 significantly enhanced the lipid content by 46.4 to 52.9%, but did not affect the growth rate of the host cells under mixotrophic culture conditions. Transcriptome profiles indicated that 1,076 transcripts were differentially regulated: of these, 754 genes were significantly upregulated and 322 genes were significantly downregulated in the transgenic strains under mixotrophic culture conditions. There are 22 significantly regulated genes (|log2 ratio| >1) involved in lipid and fatty acid metabolism. Quantitative real-time PCR and an enzyme activity assay revealed that GmDof4 significantly up-regulated the gene expression and enzyme activity of acetyl-coenzyme A carboxylase, a key enzyme for fatty acid synthesis, in transgenic C. ellipsoidea cells.ConclusionsThe hetero-expression of a transcription factor GmDof4 gene from soybean can significantly increase the lipid content but not affect the growth rate of C. ellipsoidea under mixotrophic culture conditions. The increase in lipid content could be attributed to the large number of genes with regulated expression. In particular, the acetyl-coenzyme A carboxylase gene expression and enzyme activity were significantly upregulated in the transgenic cells. Our research provides a new way to increase the lipid content of microalgae by introducing a specific transcription factor to microalgae strains that can be used for the biofuel and food industries.

Improving performance of flat-plate photobioreactors by installation of novel internal mixers optimized with computational fluid dynamics

A novel mixer was developed to improve the performance of flat-plate photobioreactors (PBRs). The effects of mixer were theoretically evaluated using computational fluid dynamics (CFD) according to radial velocity of fluid and light/dark cycles within reactors. The structure parameters, including the riser width, top clearance, clearance between the baffles and walls, and number of the chambers were further optimized. The microalgae culture test aiming at validating the simulated results was conducted indoor. The results showed the maximum biomass concentrations in the optimized and archetype reactors were 32.8% (0.89 g L(-1)) and 19.4% (0.80 g L(-1)) higher than that in the control reactor (0.67 g L(-1)). Therefore, the novel mixer can significantly increase the fluid velocity along the light attenuation and light/dark cycles, thus further increased the maximum biomass concentration. The PBRs with novel mixers are greatly applicable for high-efficiency cultivation of microalgae.