Jianhua Fan

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.

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.

The effective photoinduction of Haematococcus pluvialis for accumulating astaxanthin with attached cultivation

As the optimal source of astaxanthin, Haematococcus pluvialis was cultured for commercial production of astaxanthin through two continuous phases: cell growth and astaxanthin induction. In this study, the efficiency of an attached system for producing astaxanthin from H. pluvialis was investigated and compared to that of the suspended system (bubble column bioreactor) under various conditions. Results showed that this attached system is more suitable for photoinduction of H. pluvialis than the suspended bioreactor. Under the optimal conditions, the astaxanthin productivity of the attached system was 65.8 mg m(-2)d(-1) and 2.4-fold of that in the suspended system. This attached approach also offers other advantages over suspended systems, such as, producing astaxanthin under a wide range of light intensities and temperatures, saving water, ease to harvest cells, resisting contamination. Therefore, the attached approach can be considered an economical, environmentally friendly and highly-efficient technology for producing astaxanthin from H. pluvialis.

The effect of nutrition pattern alteration on Chlorella pyrenoidosa growth, lipid biosynthesis-related gene transcription

Heterotrophy to photoautotrophy transition leads to the accumulation of lipids in Chlorella, which has potential to produce both healthy food and biofuels. Therefore, it is of key interest to study the metabolism shift and gene expression changes that influenced by the transition. Both total and neutral lipids contents were increased rapidly within 48 h after the switch to light environment, from 24.5% and 18.0% to 35.3% and 27.4%, respectively, along with the sharp decline of starch from 42.3% to 10.4% during 24h photoinduction phase. By analyzing the correlation between lipid content and gene expression, results revealed several genes viz. me g3137, me g6562, pepc g6833, dgat g3280 and dgat g7566, which encode corresponding enzymes in the de novo lipid biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. These results represented the feasibility of lipid production through trophic converting cultivation.

Suppression subtractive hybridization reveals transcript profiling of Chlorella under heterotrophy to photoautotrophy transition.

Background Microalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. Chlorella are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in Chlorella intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear. Methodology/Principal Findings In this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs) were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1%) from the forward library and 62 (21.8%) from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense. Conclusions/Significance The data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of Chlorella from heterotrophy to photoautotrophy, which holds great potential for further improving its lipid and nutrient production.