A new mechanism for efficient hydrocarbon electro-extraction from Botryococcus braunii

In this study, various voltages, frequencies, and pulse numbers were applied to determine the extraction efficiency based on the energy spent. We also elucidated how our extraction system functions. Combining macroscopic and microscopic observations showed, under a sufficient electric field, the matrix and cells were separated, and that matrix was composed largely of hydrocarbons.

It has been proved that electric field effect on biologic material sometime come from electrostriction [28] or pressure wave [29] induced by the potential difference generated by PEF. In this respect, two hypotheses can be formulated to explain mechanism behind the extraction: (1) The retaining wall ruptures—due to movement of charged molecules, electrostriction, or pressure wave—allow cells to naturally leave the colony. (2) Polysaccharides secreted by cells and solubilized in the oily matrix are detached from cells—due to movement of charged molecules, electrostriction, or pressure wave—and having lost their anchor, cells can no longer adhere to the colony. As the retaining wall was not often observed, it is not possible to confirm the first hypothesis. Conversely, as we could see evidence of polysaccharides inside the oily matrix (Fig. 2a–c), and as detached cells leaving the colony are mostly free of oil/polysaccharides except for the cell wall (Fig. 2d; Additional file 1: Section 2-2, movies of Additional files 2, 3), there is clearly a separation between cells and oil/polysaccharide of the matrix when nsPEF are applied, supporting the second hypothesis as prevailing mechanism.

Another hypothesis to explain the mechanism behind the electric field induced cell detachment can also be considered: (3) The cells in the colony might be linked together by actin filaments [30]. Exposure to nsPEF destroys actin, loosening the link between the cells, causing them to leave the colony. Indeed, in plant cells, during phragmoplasme formation, plasmodesmata are built in such a way to connect cytoplasm and actin filaments even after the end of cell division [30]. Berghöfer et al. [31] showed that nsPEF trigger actin responses in plant cells. Positive effect of PEF on actin disassembling was also shown [32]. Further investigations should focus on actin filaments observation to validate this hypothesis, as plasmodesmata might not exist for Botryococcus braunii cells and actin might be restricted into the cytoplasm.

The extraction mechanism here is completely different from electro-extraction of other microalgae species. Other unicellular algae species, which also produce a large amount of oil, do not secrete a matrix. In those cases, the electric field must destroy the plasma membrane—or at least cause poration—in order to liberate hydrocarbon vesicles, which require higher amount of electric energy. Goettel [21] showed that 1 MJ is required to rupture cells of 1 kg of dry weight algae (Auxenochlorella protothecoides) from a suspension of near 100 g of dry weight algae per kg of suspension. The author also showed that the concentration of algae suspension did not affect efficiency [21]. In that case, 0.1 MJ was required to treat approximately one liter of algae suspension (=100 J/ml). Our treatment using two electroporation cuvettes of 450 µl (=0.9 ml) enabled extraction using 50 J (55.6 J/ml), almost with two times higher efficiency. The matrix also contains polysaccharides; therefore, the supernatant is not pure hydrocarbon and thus needs further purification. Polysaccharides hold high interest in the field of green energy as they may be used to produce bioethanol [33]; however, polysaccharides extract would be in low concentration as high percentage of the dry mass of Botryococcus braunii consists of hydrocarbons [34].

According to our screening, the best performance may be achieved at 50 kV/cm, where the lowest energy consumption was needed for extraction. Higher energy spent only marginally improves extraction, and a higher electric field would not be necessary. Also shown was that frequencies from 1 to 500 Hz have no effect on efficiency, meaning that treatment can be rapid and thus adaptable as an industrial process.

As our method with nsPEF allows use of electric fields and energies lower than the cell irreversible membrane damage threshold [35, 36], a high portion of the algae cells may survive during the extraction. This is an important advantage; as such, it may be considered an ‘in vivo extraction’ method (Fig. 7).

Current oil extraction processes are mostly destructive; after the culture reaches a stationary state, oil is extracted from an algae cake obtained by drying [37, 38]. Conversely, our method may allow the culture to restart directly after oil and polysaccharide extraction, like chemical extraction method [39]. As such, our system is adaptable as a continuous oil and polysaccharide extraction process at low energy cost. In the flow treatment process, electric field application to treat large volumes was examined for bacterial eradication and protein extraction from microalgae [9, 10, 12, 40]. In particular, it is possible to design a system using a derivation to separate algae culture from a bioreactor, treat it by nsPEF, extract oil and polysaccharide by in-line decantation (lamellar decanter might help to lead single cells on the bottom part and matrix on the upper part), and then return the medium and surviving algae to the bioreactor, as shown in Fig. 7. In such a system, production of oil and polysaccharide might be faster than in a batch system, as the culture would not have to restart from zero. After treatment, cells can directly restart to grow and divide to produce more oil and polysaccharide (Fig. 7). This type of system might also minimize the initial as well as running cost, because the separation of cells and extract can be done rapidly by simple decantation, thus requiring little energy and space.