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Progress on the fundamental research on microalgae Featured

Optimization of lipid productivity

 

Two-stage batch cultivations with P. tricornutumhave been performed to study the effect of incident light intensity on lipid production. This showed that P. tricornutumis rather sensitive to a combination of nitrogen depletion and high light intensities, leading to rapid cell death at high incident light intensities. The highest lipid yield can be obtained at relatively low light intensity combined with nitrogen depletion. The knowledge obtained during these two stage experiments was used to design a one-step lipid production process.

 

Transcriptomics

 

We have successfully implemented methods and protocols for transcriptome profiling of N-depleted and N-repletedPhaeodactylumsamples from partner WU. Mapping of the transcriptome against the annotated genome sequence from P. tricornutumrevealed that 10213 genes out of 10391 annotated genes were expressed, indicating the sufficient coverage of transcriptome. We have analysed expression levels of genes by mapping transcriptome data against gene sequences from known lipid biosynthesis (KEGG, DiatomCyc) pathways. This approach enables the identification of differentially expressed genes underlying lipid biosynthesis in P. tricornutum. This platform is thus ready to perform detailed analysis on sample series

 

Proteomics

 

 

Several protein extraction procedures from intact Phaeodactylumcells have been tested and evaluated with the aim of obtaining a maximum of protein identifications plus an optimal quantitative reproducibility of the extraction efficiency. 3 grinding methods and 2 extractions buffers have been tested. In conclusion, grinding wet sample gives maximum identifications. The 2 buffers both give high yield, with some complementarity. We are ready to perform detailed quantitative analysis on sample series.

 

Metabolomics

 

 

Several protein extraction procedures from intact Phaeodactylumcells have been tested and evaluated with the aim of obtaining a maximum of protein identifications plus an optimal quantitative reproducibility of the extraction efficiency. 3 grinding methods and 2 extractions buffers have been tested. In conclusion, grinding wet sample gives maximum identifications. The 2 buffers both give high yield, with some complementarity. We are ready to perform detailed quantitative analysis on sample series.

 

 

 

 

Proof of principle of one-step process

 

 

A strategy for one-step lipid production in P. tricornutumwas designed and tested at lab scale (1.7L flat-panel photobioreactors). Simultaneous growth and lipid accumulation was reached in nitrogen limited turbidostats operated under day/night light cycles, thereby providing a proof of principle of one-step lipid production in P. tricornutum. The fraction of TAG increased from 7 %w/w in a nitrogen replete culture to 12 %w/w in a nitrogen limited culture. Optimization of this process is needed to further increase lipid content and productivity. These experiments will be coupled to transcriptome, proteome and metabolome analysis. The combination of continuous experiments with ~omics technologies will generate fundamental insight in the mechanisms underlying lipid accumulation in P. tricornutum, and will also give directions for the cultivation strategies that will be tested outdoors.

 

We have recently defined optimal two stage starvation protocols for maximal oil productivity with Nannochloropsis, including semi-continuous cultivation with limiting nitrogen content and batch cultivation with dilution of nutrient replete culture into nutrient deficient growth media. The impact of varying salinity on both approaches was tested. While the semi-continuous cultivation gives higher oil productivity at elevated salinity, the batch cultivation gives higher oil productivity if the starvation phase is performed at lower salinity. This approach was documented by metabolomics analysis indicating that high salinity demands high concentrations of free sugars as osmoticum, which are not required under lower salinity. Those findings are now being investigated outdoors in temperature controlled panel reactors.