Extraction of EPA ethyl esters from microalgae with supercritical fluids

Useable algae components.
Useable algae components.

Recovery of algal products with supercritical fluids

Supercritical fluid extraction, a “natural and green” way of achieving product extraction, has received increased attention as an important alternative to conventional separation methods because it is simpler, faster, more efficient and avoids the consumption of large amounts of organic solvents, which are often expensive and potentially harmful. Within a project funded by the Deutsche Bundesstiftung Umwelt (DBU) a process for the recovery of polyunsaturated omega-3-fatty acids (eicosapentaenoic acid EPA; 20:5 n-3) will be developed. Depending on the algae strain, galactolipids can be extracted with ethanol from the biomass. Using transesterification, the fatty acid esters can be purified by means of supercritical CO2. So far, cell disruption is not necessary for the strain Phaeodactylum tricornutum. In order to keep the process economical without any drying step requiring high energy consumption it is also possible to use wet biomass. Furthermore, in the next step it will be necessary to study the transesterification of the galactolipids.

 

EPA from Phaeodactylum tricornutum  – alternative to EPA from fish oil

Algae produce a multitude of chemical base materials with high added value potential, such as carotenoids, fatty acids and proteins (Table ). The long-chain omega-3 fatty acid eicosapentaenoic acid (EPA, 20:5) is most often utilized as a dietary supplement. Thus far, EPA has been predominantly extracted from fish oil, where it exists in composite form with DHA (docosahexaenoic acid, 22:6). Further refinement to obtain pure EPA is a complex and expensive process. Due to the various effects of both of these fatty acids on the human body, only pure fatty acids are capable of a precise and definite application. In comparison to fish oil, many microalgae hold only EPA (ca. 5 percent by weight) along with shorter-chain fatty acids and no DHA. The goal of an ongoing project at the Institute for Interfacial Engineering IGVT at the University of Stuttgart is therefore to establish an integrated process for the production of EPA from the microalgae Phaeodactylum tricornutum as a cost-effective alternative to EPA production from fish oil.

Overview of process steps

The algal cells are cultivated with sunlight, mineral salts, and CO2 in closed photobioreactors that are developed at the Fraunhofer IGB. EPA is produced in the chloroplast membrane of the microalgae as monogalactosyldiglycerol. For extraction of the monogalactosyldiglycerols from the chloroplast membrane, different organic solvents such as ethanol have been tested. Additionally, continuous extraction via supercritical fluids has been tested. This would afford the advantage of extracting the product without potentially health-harming solvents. In order for EPA to be utilized as a dietary supplement, it must also be processed in the form of ethyl ester.

The integrated downstream processing for the production of EPA from microalgae can be broken down into multiple steps:

  • Cell disruption of the algal cells
  • Extraction of the monogalactosyldiglycerols from the
    microalgae
  • Transesterification of the monogalactosyldiglycerols to
    EPA ethyl esters
  • Further steps of purification

Results

Flowchart of the plant for the extraction with supercritical fluids.
Flowchart of the plant for the extraction with supercritical fluids.
Phases diagram of supercritical fluids using the example of CO2.
Phases diagram of supercritical fluids using the example of CO<sub>2</sub>.

Mechanical cell disruption

Because EPA is found in the chloroplast membrane of the algal cells, cell disintegration is necessary to reduce the diffusion barrier effect of the cell membrane. Here two possibilities have been tested: pressing the algal biomass with up to 2000 bar to the point of disintegrating using a high-pressure homogenizer, and using a stirred ball mill, where the cell membrane is ground up and consequently the entire cell structure is destroyed and disintegrated.

 

Extraction of galactolipids with supercritical carbon dioxide

In addition to organic solvents like ethanol, we have also employed supercritical CO2 (scCO2) for the extraction of the galactolipids from the algal cells). In the extractor, the biomass acts as a packed bed through which the scCO2 flows. The extract is subsequently isolated from the CO2 in the separator and collected. Because of the polarity of the galactolipids, the yield rate of the extraction process with scCO2 can be increased through the use of ethanol as a co-solvent. The effects of the high-pressure homogenizer versus stirred ball mill on the extraction results were also tested. It was shown here that extraction of the disintegrated biomass using the stirred ball mill method exhibited the highest yield rate of up to 85 percent.

 

Transesterification of the galactolipids to EPA ethyl esters

In the production of EPA ethyl ester for application as a dietary supplement, it is necessary to split the EPA from extracted galactolipids and to esterify it enzymatically with ethanol. For this purpose, another solvent in addition to ethanol is required in order to maintain the enzyme activity, such as scCO2. Enzymes for the transesterification are immobilized and employed as an enzyme packed bed. An ethanol extract with galactolipids is added to the continuous flow of scCO2 over the enzyme packed bed. Contingent upon the concentration of galactolipids in the ethanol extract, the residence time for complete transesterification must be adjusted correspondingly.

 

Further steps of purification and perspective

For the extraction of pure EPA ethyl esters further purification steps are required: The separation of the ethanol using rectification, polar residual components using scCO2 and the short chain fatty acid ethyl esters using scCO2-chromatography still need to be developed. To establish sustainable, resource-efficient and environmentallyfriendly processes for the algal biomass material and energy potential utilization in the future, first valuable products should be extracted from the algae and fractionated using supercritical fluids and the residual biomass could then be used for energy production, according to the principles of a biorefinery.

Project information

Project title

Integrated process for the production of omega-3 EPA in the photobioreactor using microalgae, development of disintegration and extraction processes

 

Project partners

IGVP, University of Stuttgart

 

Funding

We would like to thank the Deutsche Bundesstiftung Umwelt (DBU) for funding the project “Integrated process for the production of omega-3 EPA in the photobioreactor using microalgae, development of disintegration and extraction processes”, promotional reference 13224 – 32 at the Institute for Interfacial Engineering (IGVT) at the University of Stuttgart.