Enhanced biomass and oil production from sugarcane bagasse hydrolysate (SBH) by heterotrophic oleaginous microalga Chlorella protothecoides.

The potential use of sugarcane bagasse hydrolysate (SBH) for microalgal oil in a heterotrophic mode and the oil accumulation mechanisms by SBH-induced Chlorella protothecoides cells were investigated in this study. Results demonstrated that SBH performed better than glucose for cell growth and lipid accumulation under the same reducing sugar concentration. The lipid productivity of 0.69g/L/d was accomplished at 40g/L of reducing sugar by batch culture. Under the fed-batch culture condition, the maximum biomass and lipid productivity were 24.01g/L and 1.19g/L/d, respectively. Metabolic pathway analysis results indicated that xylose and arabinose involved in pentose phosphate pathway might be predominant over sole glucose involved in glycolysis for lipid accumulation in cells. Three metabolic checkpoints in the proposed metabolic network, including xylulose kinase, acyl-CoA dehydrogenase, and dihydrolipoyl dehydrogenase reveal new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity.

Enhanced lipid accumulation and biodiesel production by oleaginous Chlorella protothecoides under a structured heterotrophic-iron (II) induction strategy.

A structured heterotrophic-iron (II) induction (HII) strategy was proposed to enhance lipid accumulation in oleaginous Chlorella protothecoides. C. protothecoides subjected to heterotrophic-iron (II) induction achieved a favorable lipid accumulation up to 62 % and a maximum lipid productivity of 820.17 mg/day, representing 2.78-fold and 3.64-fold increase respectively over heterotrophic cultivation alone. HII-induced cells produced significantly elevated levels of 16:0, 18:1(Δ9), and 18:2(Δ9,12) fatty acids (over 90 %). The lipid contents and plant lipid-like fatty acid compositions exhibit the potential of HII-induced C. protothecoides as biodiesel feedstock. Furthermore, 31 altered proteins in HII-induced algal cells were successfully identified. These differentially expressed proteins were assigned into nine molecular function categories, including carbohydrate metabolism, lipid biosynthesis, Calvin cycle, cellular respiration, photosynthesis, energy and transport, protein biosynthesis, regulate and defense, and unclassified. Analysis using the Kyoto encyclopedia of genes and genomes and gene ontology annotation showed that malic enzyme, acyltransferase, and ACP were key metabolic checkpoints found to modulate lipid accumulation in C. protothecoides. The results provided possible applications of HII cultivation strategy in other microalgal species and new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity.

Regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime.

Proteomics in conjunction with biochemical strategy was employed to unravel regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime (HPC). Interestingly, HPC triggered transiently synthesis of starch followed by substantial lipid accumulation. And a marked decrease in intracellular protein and chlorophyll contents was also observed after 12h of photo-induction. The highest lipid content of 50.5% was achieved upon the photo-induction stage, which represented 69.3% higher than that of the end of heterotrophic cultivation. Results suggested that turnover of carbon-nitrogen-rich compounds such as starch, protein, and chlorophyll might provide carbon or energy for lipid accumulation. The proteomics analysis indicated that several pathways including glycolysis, TCA cycle, ß-oxidation of fatty acids, Calvin cycle, photosynthesis, energy and transport, protein biosynthesis, regulate and defense were involved in the lipid biosynthesis. Malate dehydrogenase and acyl-CoA dehydrogenase were suggested as key regulatory factors in enhancing lipid accumulation.

Proteomics analysis for enhanced lipid accumulation in oleaginous Chlorella vulgaris under a heterotrophic-Na⁺ induction two-step regime.

A heterotrophic-Na(+) induction (HNI) two-step regime was developed to enhance lipid accumulation in oleaginous Chlorella vulgaris. C. vulgaris was cultivated heterotrophically to a biomass of 7.8 g l(-1) in 120 h. The cells were re-suspended in fresh media supplemented with 0.5 M NaCl followed by 12 h growth to accumulate lipid to 53.4 % (w/w). The lipid productivity (625 mg l(-1) day(-1)) achieved with HNI was better than that using heterotrophy alone (405 mg l(-1) day(-1)). To promote possible applications of HNI strategy in other microalgal species, the lipid triggers and potential molecular pathways associated with lipid biosynthesis were investigated. Malic enzyme and acyl-CoA-binding protein were key metabolic checkpoints found to modulate lipid biosynthesis in cells. These results provide the foundation to develop high-lipid engineering miroalgae for industrialization of biodiesel.

Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen (N) and phosphorus (P).

This study aimed to evaluate the potential lipid accumulation of an oleaginous Chlorella protothecoides by combination cultivation mode of nitrogen (N) and phosphorus (P). Under co-deficiency of N and P, the largest lipid content (55.8%) was accomplished in C. protothecoides, which was higher than either sole P-deficiency (32.77%) or N-deficiency (52.5%), or co-repletion of N and P (control) (22.17%). However, the highest lipid productivity (224.14mg/L/day) with combination mode of N-deficiency and P-repletion represented 1.19-3.70-fold more than that of control, P-deficiency/limitation, and co-deficiency of N and P, respectively. This indicating N-deficiency plus P-repletion was a promising lipid trigger to motivate lipid accumulation in C. protothecoides cells. Further, difference gel electrophoresis (DIGE)-based proteomics was employed to reveal the molecular pathways associated with lipid biosynthesis. These results provide the foundation to develop engineering strategies targeting lipid productivity for industrial production of microalgae-based biodiesel.

Production of biomass and lipid by the microalgae Chlorella protothecoides with heterotrophic-Cu(II) stressed (HCuS) coupling cultivation.

This work for the first time investigated lipid accumulation by a two-stage regime namely heterotrophic-Cu(II) stressed (HCuS) and underlying molecular basis of lipid biosynthesis in Chlorella protothecoides cells. The results showed that the optimized biomass and lipid yield were achieved by 6.47 g/L and 5.78 g/L with this strategy. The fatty acids compositions (almost 100% of them are C15 to C20) are ideal for preparing high quality biodiesel. Further, 30 differentially expressed proteins response to HCuS were involved in carbohydrate metabolism, carbon fixation, TCA cycle, lipid metabolism, protein biosynthesis, transportation and regulation, ATP and RNA biosynthesis, nucleotide metabolism, ROS scavenging. Especially, glycolysis pathway might be the important contributor for lipid accumulation. In future, further functional analysis of these altered proteins would help to reveal more concerning lipid biosynthesis pathway.