Evaluation of Downstream Processing, Extraction, and Quantification Strategies for Single Cell Oil Produced by the Oleaginous Yeasts Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194

Abstract

Single cell oil (SCO) produced by oleaginous yeasts is considered as a sustainable source for biodiesel and oleochemicals since its production does not compete with food or feed and high yields can be obtained from a wide variety of carbon sources, e.g., acetate or lignocellulose. Downstream processing is still costly preventing the broader application of SCO. Direct transesterification of freeze-dried biomass is widely used for analytical purposes and for biodiesel production but it is energy intensive and, therefore, expensive. Additionally, only fatty acid esters are produced limiting the subsequent applications. The harsh conditions applied during direct esterification might also damage high-value polyunsaturated fatty acids. Unfortunately, universal downstream strategies effective for all yeast species do not exist and methods have to be developed for each yeast species due to differences in cell wall composition. Therefore, the aim of this study was to evaluate three industrially relevant cell disruption methods combined with three extraction systems for the SCO extraction of two novel, unconventional oleaginous yeasts, Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194, based on cell disruption efficiency, lipid yield, and oil quality. Bead milling (BM) and high pressure homogenization (HPH) were effective cell disruption methods in contrast to sonification. By combining HPH (95% cell disruption efficiency) with ethanol-hexane-extraction 46.9 ± 4.4% lipid/CDW of S. podzolica were obtained which was 2.7 times higher than with the least suitable combination (ultrasound + Folch). A. porosum was less affected by cell disruption attempts. Here, the highest disruption efficiency was 74% after BM and the most efficient lipid recovery method was direct acidic transesterification (27.2 ± 0.5% fatty acid methyl esters/CDW) after freeze drying. The study clearly indicates cell disruption is the decisive step for SCO extraction. At disruption efficiencies of >90%, lipids can be extracted at high yields, whereas at lower cell disruption efficiencies, considerable amounts of lipids will not be accessible for extraction regardless of the solvents used. Furthermore, it was shown that hexane-ethanol which is commonly used for extraction of algal lipids is also highly efficient for yeasts.

Keywords: Apiotrichum porosum DSM 27194; Saitozyma podzolica DSM 27192; cell disruption; downstream processing; lipid extraction; oleaginous yeasts; single cell oil; transesterification.

FIGURE 1

Overview of performed methods for lipid recovery. Red framed boxes were analyzed for both yeasts. The black framed methods were implemented on S. podzolica only. FAMEs, fatty acid methyl esters; FFAs, free fatty acids.

FIGURE 2

Light microscopy pictures of untreated and via BM, HPH and U mechanically disrupted, nile red stained yeast cells. (A) S. podzolica, (B) A. porosum. BM, bead mill; HPH, high pressure homogenization; U, ultrasonic treatment.

FIGURE 3

Comparison of KOH and H₂SO₄ catalysts for FAME production after HPH-BD treatment and direct transesterification of yeast biomass. % FAME per CDW from S. podzolica biomass is demonstrated in (A), while (B) presents the same for A. porosum. The standard deviation of three independent experiments are indicated by the error bars. a, b, c illustrate statistical differences (p = 0.05). FAMEs, fatty acid methyl esters; BM, bead mill; HPH, high pressure homogenization; DT, direct transesterification; CDW, cell dry weight; BD, extraction according to Bligh and Dyer.

FIGURE 4

Comparison of all processed lipid recovery methods, using BD extraction and acidic transesterification, and acidic DT method. (A) % FAME per CDW from S. podzolica biomass. (B) % FAME per CDW from A. porosum biomass. The error bars result from the standard deviation of three independent experiments. a, b, c reveal statistical differences (p = 0.05). FAMEs, fatty acid methyl esters; CDW, cell dry weight; BM, bead mill; HPH, high pressure homogenization; DT, direct transesterification; BD, extraction according to Bligh and Dyer.

FIGURE 5

Fatty acid distribution of in situ transesterified and BM prior to BD processed biomass analyzed by HPLC and GC. (A) Fatty acid profiles in% per CDW of S. podzolica. (B) Fatty acid distribution in% per CDW of A. porosum. DT, direct transesterification; BD, extraction according to Bligh and Dyer.