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This study reports on the use of pulsed electric field (PEF) as a pre-treatment step to enhance lipid extraction yield using extraction with ethanol-hexane blend on fresh oleaginous yeast Saitozyma podzolica. The yeasts were cultivated on nitrogen-depleted condition and had a lipid content of 26.4 ± 4.6% of dry weight. PEF-treatment was applied on the yeast suspension either directly after harvesting (unwashed route) or after a washing step (washed route) which induced a reduction of conductivity by a factor eight. In both cases, cell concentration was 20 g of biomass per liter of suspension. In the unwashed route, the lipid extraction efficiency increased from 7% (untreated) to 54% thanks to PEF-treatment. In case an additional washing step was added after PEF-treatment, up to 81% of the lipid content could be recovered. The washed route was even more efficient since lipid extraction yields increased from 26% (untreated) to 99% of total lipid. The energy input for the PEF-treatment never exceeded 150 kJ per liter of initial suspension. The best lipid recovery scenario was obtained using pulses of 1 μs, an electric field of 40 kV/cm and it required slightly less than 11 MJ/kgLIPID. This amount of energy can be further reduced by at least a factor five by optimizing the treatment and especially by increasing the concentration of the treated biomass. The process can be easily up-scaled and does not require any expensive handling of the biomass such as freezing or freeze-drying. These findings demonstrate the potential benefit of PEF-treatment in the downstream processing of oleaginous yeast. From a basic research point of view, the influence of conductivity on PEF energy requirements and extraction yields was examined, and results suggest a higher efficiency of PEF-treatment in terms of energy when treatment is performed at lower conductivity.

Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 hour incubation period at 25°C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kg DW . Incubation on ice remains beneficial but less efficient than at 25°C. Additionally, incubating microalgae cells in suspension at 100 g DW /L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated.

This study reports the use of hypotonic osmotic shock as a treatment step to enhance the recoveries of biofuel-convertible lipids and proteins from lipid-rich saltwater Nannochloropsis gaditana (N. gaditana) slurries (biomass content = ~140 mg biomass / g slurry, total lipid content = ~600 mg lipid /g biomass). The osmotic shock was induced through repeated washing of microalgal slurries with multiple batches of fresh water. Subjecting the slurries to 2 stages of freshwater washing resulted in a measurable damage to cell membranes (the uptake of membrane permeability marker increased by 6 folds), a partial loss of cell viability (only 64% of available cells were recoverable), and a minor release of free protein (~2 wt% of available protein) from the biomass into the interstitial space of the slurries. Hypotonic osmotic shock was revealed to be ineffective in rupturing N. gaditana slurries (only 13 ± 9% of available cells were ruptured after 2-stage washing) and, as such, had a limited prospect as a stand-alone cell disruption technology for the saltwater strain. The washing treatment, however, was found to be able to weaken the structural integrity of N. gaditana slurries and enhance the performance of subsequent mechanical or chemical cell disruption technologies when installed as a preparatory step. Applying the washing treatment prior to high-pressure homogenisation (HPH) and low-solvent-to-biomass ratio hexane extraction (hexane: slurry = 1:1 w/w) for the recovery of biofuel-convertible lipids increased the extent of cell rupture from 28 ± 8 to 46 ± 19% of available cells and more than doubled neutral lipid yield from 25.1 ± 2.0 to 64.6 ± 4.9 wt% of available neutral lipid. Initial analysis revealed that the washing treatment had a minimal energy cost (~6% of the total energy expenditure of downstream processing) and that its integration into HPH + hexane lipid recovery led to a 2.5 fold increase in the energy output of the biomass. Partnering the washing treatment with NaOH hydrolysis increased protein yield from 6.7 ± 2.4 to 31.9 ± 10.7 wt% of available protein.