Pulsed Electric Field Treatment Promotes Lipid Extraction on Fresh Oleaginous Yeast Saitozyma podzolica DSM 27192

Abstract

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/kg_LIPID. 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.

Keywords: electroporation; fresh biomass; lipid; oleaginous yeast; pulsed electric field; pulsed electric field assisted extraction; solvent extraction.

FIGURE 1

Photos of the PEF-treatment chambers. (A) Disassembled treatment chambers. The chambers are designed for treating suspensions with conductivities of 1.5 mS/cm (left) and 12 mS/cm (right) with a 50 Ohm transmission-line generator. (B) Illustration of an assembled treatment chamber with the tubes used for the continuous flow of the yeast suspension.

FIGURE 2

Yeast cultivation. (A) Growth curve of S. podzolica on mineral salt medium and daily fed glucose, ammonium consumption, biomass (CDW) and lipid production (indirectly as FAME/CDW) are indicated as average ± standard deviation of four independent cultivations. (B) Composition of the yeast on harvesting day. Average + standard deviation of four independent cultivations.

FIGURE 3

Schematic of the two treatment paths “Unwashed” and “Washed”.

FIGURE 4

Impact of washing on lipid extraction (A) and Yo-Pro uptake (B). Two different washing techniques were implemented. In technique A, distilled water was added to reduce conductivity in yeast broth prior to centrifugation and in technique B, the yeast suspension was centrifuged beforehand, subsequently the biomass was resuspended to the same concentration as before with distilled water supplemented with NaCl to reach the targeted conductivity. Results are average ± standard deviation of two independent experiments (n = 2) with duplicates in each. Control refer to yeast processed directly after harvest i.e., not submitted to any washing treatment. Statistical significances are indicated in comparison with control.

FIGURE 5

Lipid extraction yields obtained after processing along the “unwashed-route”. PEF-treatment was applied directly after harvesting. Pulse duration was kept constant at Δt = 1 μs. (A) Impact of the electric field value at a constant specific treatment energy of 150 kJ/L. The x-axis indicates for each condition the applied electric field in kV/cm (upper values), the repetition rate in Hz at which pulses were applied (middle values) and the working flow rate of the suspension in the treatment chambers (lower values). (B) Impact of the specific treatment energy at a constant electric field value of 14 kV/cm. The applied specific energies, the repetition rate in Hz at which pulses were applied as well as the working flow rates have been indicated on the x-axis. For both graphics, the gray bars are the results of lipid extraction performed directly after the PEF-treatment (U) and the green bars are the lipid extraction yields obtained when a washing step is added after PEF-treatment before addition of extraction solvents (W). The yellow lines indicate the average ± standard deviation of Soxhlet extractions, as absolute control. Results are reported as the average + standard deviation of three independent experiments (n = 3) with samples processed in duplicates. Statistical significances are indicated in comparison with the unwashed control.

FIGURE 6

Strategy to compare “washed-route” with the “unwashed-route.” In the washed route the conductivity of the yeast suspension is 8 times lower than in the “unwashed-route”.

FIGURE 7

Lipid extraction yields obtained after processing along the “washed-route.” PEF-treatment was applied on the yeast washed after harvesting. Impact of the specific treatment energy at a constant electric field value of (A) 14 kV/cm and (B) 40 kV/cm. For the bottom graph, the gray bars are the results of lipid extraction performed directly after the PEF-treatment and the green bars are the lipid extraction yields obtained when a washing step is added after PEF-treatment before addition of extraction solvents. The yellow lines illustrate the average ± standard deviation of Soxhlet extractions, as absolute control. For both experiments, the results are reported as the average + standard deviation of three independent experiments (n = 3) with samples processed in duplicates. Statistical significances are indicated in comparison with the unwashed control.