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 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kg. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100g/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.

Background Microalgae have attracted considerable interest due to their ability to produce a wide range of valuable compounds. Pulsed Electric Fields (PEF) has been demonstrated to effectively disrupt the microalgae cells and facilitate intracellular extraction. To increase the commercial viability of microalgae, the entire biomass should be exploited with different products extracted and valorized according to the biorefinery scheme. However, demonstrations of multiple component extraction in series are very limited in literature. This study aimed to develop an effective lipid extraction protocol from wet Scenedesmus almeriensis after PEF-treatment with 1.5 MJ·kgDW−1. A cascade process, i.e., the valorization of several products in row, was tested with firstly the collection of the released carbohydrates in the water fraction, then protein enzymatic hydrolysis and finally lipid extraction. Biomass processed with high pressure homogenization (HPH) on parallel, served as benchmark. Results Lipid extraction with ethanol:hexane (1:0.41 vol/vol) offered the highest yields from the different protocols tested. PEF-treatment promoted extraction with almost 70% of total lipids extracted against 43% from untreated biomass. An incubation step after PEF-treatment, further improved the yields, up to 83% of total lipids. Increasing the solvent volume by factor 2 offered no improvement. In comparison, extraction with two other systems utilizing only ethanol at room temperature or elevated at 60 °C were ineffective with less than 30% of total lipids extracted. Regarding cascade extraction, carbohydrate release after PEF was detected albeit in low concentrations. PEF-treated samples displayed slightly better kinetics during the enzymatic protein hydrolysis compared to untreated or HPH-treated biomass. The yields from a subsequent lipid extraction were not affected after PEF but were significantly increased for untreated samples (66% of total lipids), while HPH displayed the lowest yields (~ 49% of total lipids). Conclusions PEF-treatment successfully promoted lipid extraction from S. almeriensis but only in combination with a polar:neutral co-solvent (ethanol:hexane). After enzymatic protein hydrolysis in cascade processing; however, untreated biomass displayed equal lipid yields due to the disruptive effect of the proteolytic enzymes. Therefore, the positive impact of PEF in this scheme is limited on the improved reaction kinetics exhibited during the enzymatic hydrolysis step.

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Pulsed Electric Fields (PEF) is a known technique for the permeabilization of cell membranes, which can considerably foster intracellular component extraction from microalgae. During this phenomenon, the cells are subjected to short electrical pulses leading to the deconstruction of the cell membrane. However, it is currently uncertain in what way, if any, the microalgae cell wall is affected during pulsing. In this study, freshly harvested Auxenochlorella protothecoides (AP) and Chlorella vulgaris (CV) were subjected to PEF treatment with an energy input of 1.5MJ per kilogram of dry matter and then fed into a High Pressure Homogenizer (HPH) for 5 passes at 1500 bar. The percentage of intact cells after each pass was determined and compared with Control biomass that underwent the same homogenization. AP and CV autotrophic had almost 40% intact cells at the end of homogenizing whereas AP mixotrophic 20%. In all cases, no major difference on the disruption degree of pulsed and control samples was observed, indicating that the resistance to mechanical stress of the cell, a function of the cell wall, is not affected by PEF. Scanning Electron Microscopy (SEM) observation of the cells also showed no superficial or structural cell alteration after pulsation.

Pulsed electric field treatment performed on fresh yeast biomass induced permeabilisation of cells and greatly improved the yields of subsequent lipid extraction using ethanol and hexane. of oil raises the competition between food and raw materials production, since agricultural land is limited (Lee and Lavoie, 2013). Into the bargain, an expansion of oil seed cultures would lead to forest land destruction (Escobar et al., 2009). Microbial oils, known as single cell oils (SCO), produced by yeast, microalgae, fungi, and bacteria may overcome all these challenges and act as potential feedstock for crude and plant oil for various applications such as fuels, additives for food and cosmetics, and building blocks for oleochemicals (Ochsenreither et al., 2016; Probst et al., 2016; Vasconcelos et al., 2019). One of the most promising oleaginous microorganisms are yeasts, since they are able to accumulate more than 70% of their cell dry weight (CDW) of lipids (Ratledge, 1991; Probst et al., 2016). The production of SCO by yeasts is independent of season, climate, and location and requires limited amounts of area for cultivation. Additionally, high growth rate and oil productivity are achieved due to short duplication time and the possibility to up-scale cultivation processes, which renders an industrial use realistic (Li et al., 2008; Ageitos et al., 2011). Furthermore, unconventional carbon sources, e.g., lignocellulosic material and waste from food and other industries can be metabolized, enabling waste recycling and guaranteeing a sustainable process